Mucin hypersecretion inhibitors and methods of use

ABSTRACT

Peptides are provided that comprise less than 24 amino acids. The peptides have an amino acid sequence selected from the group consisting of: (a) an amino acid sequence having from 4 to 6 contiguous amino acids of a reference sequence PEPTIDE 1; (b) an amino acid sequence substantially identical to the sequence defined in (a); and (c) a variant of the amino acid sequence defined in (a). Also provided is a non-myristoylated MANS peptide. Various methods of using the peptides are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application under 37 C.F.R. §1.53(b) ofpending patent application Ser. No. 11/335,564 filed Jan. 20, 2006, nowU.S. Pat. No. 7,524,926, issued on Apr. 28, 2009, and which also is aContinuation of PCT Application No. PCT/US2006/002032 filed on Jan. 20,2006, and both of which claim benefit of Provisional Application No.60/645,293, filed Jan. 20, 2005, all of which are herein incorporated intheir entirety by reference.

BACKGROUND OF THE INVENTION

The invention generally relates to compositions comprising peptides andmethods for their use.

FIELD OF THE INVENTION

Mucus is a biological liquid that is capable of forming gels. It is amixture of components, including water and secretory products from avariety of cells. Mucins, also called mucus glycoproteins or epithelialglycoproteins, are a major component of mucus and are glycoconjugatescharacterized by numerous oligosaccharide side chains linked to apeptide core by N- and O-linkages. Hypersecretion of mucin (theglycoprotein component of mucus) occurs in several respiratory diseasesincluding asthma, chronic bronchitis, and cystic fibrosis (CF), and is arisk factor for mortality in patients with these diseases.

In the airways, mucins are released onto the airway surface from gobletcells in the surface epithelium, and from mucus cells of submucosalglands. The total amount of surface liquid (mucus) in the airways is theresult of the rate of mucus secretion in conjunction with the rate ofclearance of mucus (by epithelial reabsorption, evaporation, ciliarytransport, and cough transport), i.e., the result of a differencebetween the rate of mucus secretion and the rate of clearance of mucus.Under “normal” conditions, the rate of secretion and clearance of mucusare balanced so that only a thin surface layer of liquid covers thetracheobronchial tree. Mucus hypersecretion (if not accompanied by aconcomitant increase in mucus clearance) results in a net increase inthe amount of mucous relative to normal conditions and leads toaccumulation of airway mucus, which can result in airflow obstructionand increased retention of inhaled particulate and microbial matter.

Hypersecretion of mucus contributes to the pathogenesis of a largenumber of airway inflammatory diseases in both humans and non-humananimals. Increased mucus secretion is seen in chronic disease statessuch as asthma, chronic obstructive pulmonary disease (COPD) and chronicbronchitis; in genetic diseases such as cystic fibrosis; in allergicconditions (atopy, allergic inflammation); in bronchiectasis; and in anumber of acute, infectious respiratory illnesses such as pneumonia,rhinitis, influenza, and the common cold.

Accompanying hypersecretion of mucus in many of these respiratorydiseases is the increased presence of inflammatory cells in the airways.These cells contribute greatly to the pathology of these diseases viathe tissue damage and destruction done by the inflammatory mediatorsreleased from these cells. One example of such destruction via thischronic inflammation occurs in cystic fibrosis patients where mediatorsreleased from neutrophils (i.e. myeloperoxidase) induce the desquamationof the airway epithelial tissue.

Mammalian airways are lined by a thin layer of mucus produced andsecreted by airway epithelial (goblet) cells and submucosal glands. Indiseases such as asthma, COPD, chronic bronchitis, and cystic fibrosis,hypersecretion of mucus is a common lesion. Excess mucus can contributeto obstruction, susceptibility to infection, and even to destruction ofairway walls and contiguous tissues. The major components of mucus aremucin glycoproteins synthesized by secretory cells (i.e., goblet cellsand mucus cells) and stored within cytoplasmic membrane-bound granules.Mucins are a family of glycoproteins secreted by the epithelial cellsincluding those at the respiratory, gastrointestinal and femalereproductive tracts. Mucins are responsible for the viscoelasticproperties of mucus and at least eight mucin genes are known. See U.S.patent application Ser. No. 10/180,753 (Publication No. U.S.2003/0013652). Mucociliary impairment caused by mucin hypersecretionand/or mucus cell hyperplasia leads to airway mucus plugging thatpromotes chronic infection, airflow obstruction and sometimes death.Many airway diseases such as chronic bronchitis, chronic obstructivepulmonary disease, bronchiectacis, asthma, cystic fibrosis and bacterialinfections are characterized by mucin overproduction. See U.S. patentapplication Ser. No. 10/180,753 (Publication No. U.S. 2003/0013652).Upon appropriate stimulation, mucin granules are released via anexocytotic process in which the granules translocate to the cellperiphery where the granule membranes fuse with the plasma membrane,allowing for luminal secretion of the contents.

Despite the obvious pathophysiological importance of this process,intracellular signaling mechanisms linking stimulation at the cellsurface to mucin granule release have only recently been elucidated. SeeLi et al., Journal of Biological Chemistry, 276: 40982-40990 (2001). Themyristoylated, alanine-rich C kinase substrate (MARCKS) protein isbelieved to be required for mucus secretion by human bronchialepithelial cells. It has been hypothesized that MARCKS binds, atdifferent sites, to secretory granule membranes and to the actincytoskeleton to serve as a physical link between the contractilecytoskeleton and mucin granules, and could have a role in guidingsecretory granules to docking sites on the cell membrane. See Singer etal., “A MARCKS-related peptide blocks mucus hypersecretion in a mousemodel of asthma”, Nature Medicine, 10: 193-196 (2004). MANS peptide(myr-peptide 1) is a myristoylated N-terminal 24 amino acid sequence ofa protein called the “Myristoylated Alanine Rich C-Kinase Substrate”which is normally abbreviated as MARCKS protein. A 0.24 amino-acidfragment of MARCKS, myristoylated N-terminal sequence (MANS) peptide,has been shown to inhibit mucin release in vitro and has also been shownto block mucus hypersecretion in a mouse model of asthma. See Li et al.and Singer et al., supra.

The importance of myristoylation to promote translocation of peptidesacross membranes through the lipid bilayer is known. A recent studydemonstrated this importance by showing that non-myristoylated peptidesdo not get through the cell membrane as compared to myristoylatedpeptides. See A. Harishchandran et al., “Interaction of aPseudosubstrate Peptide of Protein Kinase C and its Myristoylated Formwith Lipid Vesicles. Only the Myristoylated Form Translocates into LipidBilayer,” Biochem. Biopys. Acta, 1713: 73-82 (2005).

SUMMARY OF THE INVENTION

In one aspect, a peptide is provided that consists of less than 24 aminoacids and has an amino acid sequence selected from the group consistingof: (a) an amino acid sequence having from 4 to 23 contiguous aminoacids of a reference amino acid sequence defined as PEPTIDE 1, which isalso known as the MANS peptide, and (b) an amino acid sequencesubstantially identical to the amino acid sequence defined in (a). Oneor more amino acids of the peptide are optionally independentlychemically modified, and the peptide has a mucin-inhibiting effect whenadministered to a mammal in a mucin-inhibiting amount.

In another aspect, a peptide is provided that consists of less than 24amino acids and has an amino acid sequence selected from the groupconsisting of: (a) an amino acid sequence having from 4 to 23 contiguousamino acids of a reference amino acid sequence defined as PEPTIDE 1; and(b) an amino acid sequence substantially identical to the sequencedefined in (a). The N-terminal and C-terminal amino acids of the peptideare optionally independently chemically modified. The peptide has amucin-inhibiting effect when administered to a mammal in amucin-inhibiting amount and has a greater mucin-inhibiting effect on amammal than MANS peptide when administered at equal concentrations.

In a further aspect, a peptide is provided that consists of less than 24amino acids and has an amino acid sequence selected from the groupconsisting of: (a) an amino acid sequence having from 4 to 23 contiguousamino acids of a reference amino acid sequence defined as PEPTIDE 1; and(b) an amino acid sequence substantially identical to the sequencedefined in (a). The N-terminal and C-terminal amino acids of the peptideare optionally independently chemically modified. The peptide hasgreater aqueous solubility than MANS peptide and has a mucin-inhibitingeffect when administered to a mammal in a mucin-inhibiting amount.

In yet another aspect, a method of inhibiting mucin hypersecretion in amammal is provided. The method comprises administering to the mammal amucin-inhibiting amount of a peptide that inhibits mucin secretion. Thepeptide consists of less than 24 amino acids and has an amino acidsequence selected from the group consisting of: (a) an amino acidsequence having from 4 to 23 contiguous amino acids of a reference aminoacid sequence defined as PEPTIDE 1; and (b) an amino acid sequencesubstantially identical to the sequence defined in (a). One or moreamino acids of the peptide are optionally independently chemicallymodified.

In a further aspect, a method of inhibiting mucin hypersecretion in amammal is provided. The method comprises administering to the mammal amucin-inhibiting amount of a peptide that inhibits mucin secretion. Thepeptide consists of less than 24 amino acids and has an amino acidsequence selected from the group consisting of: (a) an amino acidsequence having from 4 to 23 contiguous amino acids of a reference aminoacid sequence defined as PEPTIDE 1; and (b) an amino acid sequencesubstantially identical to the sequence defined in (a). The N-terminaland C-terminal amino acids of the peptide are optionally independentlychemically modified, and the peptide has a greater mucin-inhibitingeffect on a mammal than MANS peptide when administered at equalconcentrations.

In yet a further aspect, a method of inhibiting mucin hypersecretion ina mammal is provided. The method comprises administering to the mammal amucin-inhibiting amount of a peptide that inhibits mucin secretion. Thepeptide consists of less than 24 amino acids and has an amino acidsequence selected from the group consisting of: (a) an amino acidsequence having from 4 to 23 contiguous amino acids of a reference aminoacid sequence defined as PEPTIDE 1; and (b) an amino acid sequencesubstantially identical to the sequence defined in (a). The N-terminaland C-terminal amino acids of the peptide are optionally independentlychemically modified, and the peptide has greater aqueous solubility thanMANS peptide.

In another aspect, a peptide is provided that consists of less than 24amino acids and has an amino acid sequence consisting of a variant of anamino acid sequence having from 4 to 23 contiguous amino acids of areference amino acid sequence defined as PEPTIDE 1. The N-terminal andC-terminal amino acids of the peptide are optionally chemicallymodified. The peptide has a mucin-inhibiting effect when administered toa mammal in a mucin-inhibiting amount, has greater aqueous solubilitythan MANS peptide, and has a greater mucin-inhibiting effect on a mammalthan MANS peptide when administered at equal concentrations.

The peptides of the current invention are useful to reduce mucinhypersecretion and/or inhibit (i.e., reduce to normal levels or to lessthan normal levels) mucin hypersecretion in the treatment of diseasesand in the treatment of disease symptoms in which mucin hypersecretionis exhibited such as is seen in chronic disease states such as asthma,chronic obstructive pulmonary disease (COPD) and chronic bronchitis; ingenetic diseases such as cystic fibrosis; in allergic conditions (atopy,allergic inflammation); in bronchiectasis; and in a number of acute,infectious respiratory illnesses such as pneumonia, rhinitis, influenza,and the common cold.

In a further embodiment, a peptide is provided that consists of asequence selected from the group consisting of: (a) an amino acidsequence having the sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1);and (b) an amino acid sequence substantially identical to the sequencedefined in (a); wherein the N-terminal amino acid of the peptide is notmyristoylated and the C-terminal amino acid of the peptide is optionallyindependently chemically modified, the peptide having a mucinhypersecretion-inhibiting effect when administered to a mammal in amucin hypersecretion-inhibiting amount. This peptide is useful fortreating mucus hypersecretion in pulmonary diseases.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to methods and compositions for varioususes, including the inhibition of mucin hypersecretion (i.e., theinhibition of mucin release) and mucus production (sometimes referred toherein as inhibition of mucus secretion) in a mammal. Prior todescribing this invention in further detail, however, the followingterms will first be defined.

DEFINITIONS

“Mucin-inhibiting effect”, “mucin-inhibiting activity”, or “inhibitingmucin secretion” means a reduction in the amount of mucin secretion(i.e., mucin release), and does not necessarily mean the completecessation of mucin secretion. Administration of a composition having amucin-inhibiting effect results in decreased mucin secretion compared tothat which would occur, or would be expected, in the absence of suchcomposition. In one aspect, the amount of the decrease in mucinsecretion may be from about 5% of the amount which is secreted orhypersecreted above normal levels to about 100% of the amount which issecreted or hypersecreted above normal levels of secretion. In anotheraspect, the amount of the decrease in mucin secretion may be from about5% of the amount which is secreted or hypersecreted above normal levels(i.e., from about 5% of the amount secreted above normal levels) to anamount which is below normal level of secretion, such as to about 50% ofthe amount secreted at normal levels of secretion.

“Mucus-inhibiting effect”, “mucus-inhibiting activity”, or “inhibitingmucus production” means a reduction in the amount of mucus production,and does not necessarily mean the complete cessation of mucusproduction. Administration of a composition having a mucus-inhibitingeffect results in decreased mucus production compared to that whichwould occur, or would be expected, in the absence of such composition.

“Mucin-inhibiting amount” of a composition is that amount that reducesor inhibits mucin secretion (i.e., mucin release) as compared to thatwhich would occur in the absence of the composition, such as an amountwhich reduces mucin secretion from about 5% to about 100% of the amountof mucin which is hypersecreted above normal levels.

“Mucus-inhibiting amount” of a composition is that amount that reducesor inhibits mucus production as compared to that which would occur inthe absence of the composition.

In the reference peptide, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1), atthe N-terminal position of the reference peptide, G is at position 1;adjacent to G at position 1 is A at position 2; adjacent to A atposition 2 is Q at position 3; adjacent to Q at position 3 is F atposition 4; adjacent to F at position 4 is S at position 5; adjacent toS at position 5 is K at position 6; adjacent to K at position 6 is T atposition 7; adjacent to T at position 7 is A at position 8; adjacent toA at position 8 is A at position 9; adjacent to A at position 9 is K atposition 10; adjacent to K at position 10 is G at position 11; adjacentto G at position 11 is E at position 12; adjacent to E at position 12 isA at position 13; adjacent to A at position 13 is A at position 14;adjacent to A at position 14 is A at position 15; adjacent to A atposition 15 is E at position 16; adjacent to E at position 16 is R atposition 17; adjacent to R at position 17 is P at position 18; adjacentto P at position 18 is G at position 19; adjacent to G at position 19 isE at position 20; adjacent to E at position 20 is A at position 21;adjacent to A at position 21 is A at position 22; adjacent to A atposition 22 is V at position 23; and adjacent to V at position 23 is Aat position 24, wherein position 24 is the C-terminal position of thereference peptide.

A “variant” of a reference peptide or a variant of a 4 to 23 amino acidsegment of a reference peptide is a peptide which has an amino acidsequence which differs from the amino acid sequence of the referencepeptide or from the amino acid sequence of the segment of the referencepeptide, respectively, in at least one amino acid position in thereference peptide or reference peptide segment amino acid sequence,respectively, but which retains mucin- or mucus-inhibiting activity,which activity is typically between 0.1 to 10 times the activity of thereference peptide or segment, respectively, preferably between 0.2 to 6times the activity of the reference peptide or segment, respectively,more preferably between 0.3 to 5 times the activity of the referencepeptide or segment, respectively. A “variant” of a reference amino acidsequence or a variant of a 4 to 23 amino acid segment of a referenceamino acid sequence is an amino acid sequence that differs by at leastone amino acid from the reference amino acid sequence or from thesegment of the reference amino acid sequence, respectively, but has anamino acid sequence of a peptide that retains mucin- or mucus-inhibitingactivity of the peptide or segment, respectively, encoded by thereference amino acid sequence, which activity is typically between 0.1to 10 times the activity of the peptide or segment, respectively, of thereference sequence, preferably between 0.2 to 6 times the activity ofthe peptide or segment of the reference sequence, respectively, morepreferably between 0.3 to 5 times the activity of the peptide or segmentof the reference sequence, respectively. A substitution variant peptideor a substitution variant amino acid sequence may vary (i.e., differ)from a reference peptide or reference amino acid sequence by one or moreamino acid substitutions in the reference amino acid sequence; adeletion variant peptide or a deletion variant amino acid sequence mayvary (i.e., differ) from a reference peptide or reference amino acidsequence by one or more amino acid deletions in the reference amino acidsequence; and an addition variant peptide or an addition variant aminoacid sequence may vary (i.e., differ) from a reference peptide sequenceor reference amino acid sequence by one or more amino acid additions inthe reference sequence. A variant peptide or variant amino acid sequencecan result from a substitution of one or more amino acids (e.g.,substitution of at least 1, 2, 3, 4, 5, 6, 7, or 8 amino acids) in areference sequence, or can result from a deletion of one or more aminoacids (e.g., deletion of at least 1, 2, 3, 4, 5, 6, 7, or 8 amino acids)in a reference sequence, or can result from an addition of one or moreamino acids (e.g., addition of at least 1, 2, 3, 4, 5, 6, 7, or 8 aminoacids) in a reference sequence, or a combination thereof in any order. Asubstitution variant 4 to 23 amino acid peptide segment or asubstitution variant 4 to 23 amino acid segment sequence may vary (i.e.,differ) from a reference 4 to 23 amino acid peptide segment or reference4 to 23 amino acid segment sequence by one or more amino acidsubstitutions in the reference amino acid segment sequence; a deletionvariant 4 to 23 amino acid peptide segment or a 4 to 22 amino aciddeletion variant amino acid segment sequence may vary (i.e., differ)from a 5 to 23 reference peptide segment or a 5 to 23 amino acidreference amino acid segment sequence by one or more amino aciddeletions in the reference amino acid segment sequence; and an 4 to 23amino acid addition variant peptide or a 4 to 23 amino acid additionvariant amino acid sequence may vary (i.e., differ) from a 4 to 22 aminoacid reference peptide sequence or a 4 to 22 amino acid reference aminoacid sequence by one or more amino acid additions in the referencesequence. A 4 to 23 amino acid variant peptide or a 4 to 23 amino acidvariant amino acid sequence can result from a substitution of one ormore amino acids (e.g., substitution of at least 1, 2, 3, 4, 5, 6, 7, 8amino acids) in a 4 to 23 amino acid segment of a reference amino acidsequence, or can result from a deletion of one or more amino acids(e.g., deletion of at least 1, 2, 3, 4, 5, 6, 7, or 8 amino acids) in arespectively larger reference amino acid sequence, or can result from anaddition of one or more amino acids (e.g., addition of at least 1, 2, 3,4, 5, 6, 7, or 8 amino acids) in a respectively smaller reference aminoacid sequence, or from a combination thereof. Preferably, a variantpeptide or amino acid sequence varies from a reference peptide or from asegment of a reference peptide or from a reference amino acid sequenceor from a segment of a reference amino acid sequence, respectively, byless than 10 amino acid substitutions, deletions, and/or additions; morepreferably less than 8 amino acid substitutions, deletions, and/oradditions; even more preferably less than 6 amino acid substitutions,deletions, and/or additions; and even more preferably less than 5 aminoacid substitutions, deletions, and/or additions; and yet even morepreferably less than 4 amino acid substitutions, deletions, and/oradditions. Most preferably the variant amino acid sequence differs froma reference peptide or segment amino acid sequence by one or two orthree amino acids.

“Sequence identity” means, with respect to amino acid sequences of twopeptides, the number of positions with identical amino acids divided bythe number of amino acids in the shorter of the two sequences.

“Substantially identical” means, with respect to comparison of the aminoacid sequences of two peptides or comparison of the amino acid sequencesof two peptide segments (e.g. segments of a reference peptide amino acidsequence), that the amino acid sequence of the peptides or segments ofpeptides have at least 75% sequence identity, preferably at least 80%sequence identity, more preferably at least 90% sequence identity, andmost preferably at least 95% sequence identity.

The term “peptide” as used herein includes the peptide as well aspharmaceutically acceptable salts of the peptide.

An “isolated” peptide, as used herein, means a naturally-occurringpeptide that has been separated or substantially separated from thecellular components (e.g., nucleic acids and other peptides) thatnaturally accompany it by purification, recombinant synthesis, orchemical synthesis, and also encompasses non-naturally-occurringrecombinantly or chemically synthesized peptides that have been purifiedor substantially purified from cellular components, biologicalmaterials, chemical precursors, or other chemicals.

The following three-letter and one-letter amino acid abbreviations areused throughout the text: Alanine: (Ala) A; Arginine: (Arg) R;Asparagine: (Asn) N; Aspartic acid: (Asp) D; Cysteine: (Cys) C;Glutamine: (Gln) Q; Glutamic acid: (Glu) E; Glycine: (Gly) G; Histidine:(His) H; Isoleucine: (Ile) I; Leucine: (Leu) L; Lysine: (Lys) K;Methionine: (Met) M; Phenylalanine: (Phe) F; Proline: (Pro) P; Serine:(Ser) S; Threonine: (Thr) T; Tryptophan: (Tip) W; Tyrosine: (Tyr) Y;Valine: (Val) V. Additional three letter symbols of amino acids usefulherein include, in brackets, (Hyp) for hydroxyproline, (Nle) fornorleucine, (Orn) for ornithine, (Pyr) for pyroglutamic acid and (Sar)for sarcosine. By convention, the amino (or N-terminal) end of a peptideappears at the left end of a written amino acid sequence of the peptideand the carboxy (or C-terminal) end appears at the right end of awritten amino acid sequence. The amino acid sequence of a peptide can bewritten in single letter symbols to represent the amino acids which arecovalently linked by peptide amide bonds in the peptide.

Table IX contains a list of amino acid sequences in single letterabbreviation format together with a respectively corresponding peptidenumber and SEQ ID NO. The reference peptide amino acid sequence islisted as peptide 1. Amino acid sequences of peptides of the inventionhaving an amino acid sequence of from 4 to 23 contiguous amino acids ofthe reference amino acid sequence are listed in as peptides 2 to 231,together with the amino acid sequence of a random N-terminal sequence(RNS) comprising amino acids of the MANS peptide as peptide 232. Aminoacid sequences of representative variants of amino acid sequences ofpeptides of the invention as described herein are also listed aspeptides 233 to 245 and 247 to 251. This variant peptides listed are notintended to be a limiting group of peptides, but are presented only toserve as representative examples of variant peptides of the invention.Also presented is a representative reverse amino acid sequence and arepresentative random amino acid sequence of peptide of the invention.The reverse and random amino acid sequences in the table are notintended to be representative of the invention.

An amino acid sequence of a peptide listed in Table IX can be chemicallymodified. For example, if an amino acid sequence of a peptide listed inTable IX is chemically modified at the N-terminal amine to form an amidewith a carboxylic acid, the resulting peptide is sometimes referred toherein by a combination of an identifier for the carboxylic acid as aprefix linked by a hyphen to the peptide number. For example, withrespect to peptide 79 as an example, an N-terminal myristoylated peptide79 may sometimes be referred to herein as “myristoylated-peptide 79” or“myr-peptide 79”; an N-terminal acetylated peptide 79 may sometimes bereferred to herein as “acetyl-peptide 79” or “Ac-peptide 79”. A cyclicversion of peptide 79 may be referred to as “cyclic-peptide 79” or“eye-peptide 79”. Also, for example, if an amino acid sequence of apeptide listed in Table IX is chemically modified at the C-terminalcarboxylic group, for example by an amine such as ammonia to form aC-terminal amide, the resulting peptide is sometimes referred to hereinby a combination of an identifier for the amine residue as a suffixlinked by a hyphen to the peptide number. Thus, for example, aC-terminal amide of peptide 79 can be sometimes referred to as“peptide-NH₂”. When the N-terminal amine of the peptide (e.g., peptide79) is chemically modified by, for example, a myristoyl group and theC-terminal carboxylic group is chemically modified by, for example, anammonia group to form an amide as above, the resulting peptide can besometimes referred to, using both prefix and suffix notation, as“myr-peptide 79-NH₂”.

The invention involves peptides having amino acid sequences comprisingless than 24 amino acids with amino acid sequences related to the aminoacid sequence of MANS peptide (i.e., the MANS peptide ismyristoyl-PEPTIDE 1 and the reference 24-amino acid sequence of the MANSpeptide is PEPTIDE 1). The peptides of the current invention consist ofamino acid sequences containing less than 24 amino acids, and mayconsist of from 8 to 14, from 10 to 12, from 9 to 14, from 9 to 13, from10 to 13, from 10 to 14, at least 9, at least 10, or the like aminoacids. The peptides are typically straight chains, but may be cyclicpeptides as well. In addition, the peptides may be isolated peptides.

With respect to PEPTIDE 1, the reference 24 amino acid sequence, asegment of 23 continuous amino acids of the reference amino acidsequence is sometimes referred to herein as a 23-mer. Analogously, asegment of 22 continuous amino acids of the reference sequence issometimes referred to herein as a 22-mer; a 21 amino acid sequence as a21-mer; a 20 amino acid sequence as a 20-mer; a 19 amino acid sequenceas a 19-mer; an 18 amino acid sequence as an 18-mer; a 17 amino acidsequence as a 17-mer; a 16 amino acid sequence as a 16-mer; a 15 aminoacid sequence as a 15-mer; a 14 amino acid sequence as a 14-mer; a 13amino acid sequence as a 13-mer; a 12 amino acid sequence as a 12-mer;an 11 amino acid sequence as an 11-mer; a 10 amino acid sequence as a10-mer; a 9 amino acid sequence as a 9-mer; an 8 amino acid sequence asan 8-mer; a 7 amino acid sequence as a 7-mer; a 6 amino acid sequence asa 6-mer; a 5 amino acid sequence as a 5-mer; and a 4 amino acid sequenceas a 4-mer. In one aspect, any of these “4- to 23-mer” amino acidsequences, which are themselves peptides (sometimes herein denoted asH₂N-peptide-COOH), can be independently chemically modified, forexample, by chemical modification, which chemical modification can beselected from the group consisting of (i) amide formation at theN-terminal amine group (H₂N-peptide-) such as with, for example, a C1 orpreferably with a C2 (acetic acid) to C22 carboxylic acid; (ii) amideformation at the C-terminal carboxylic group (-peptide-COOH) such aswith, for example, ammonia or with a C1 to C22 primary or secondaryamine; and (iii) a combination of thereof.

The peptides have an amino acid sequence selected from the groupconsisting of (a) an amino acid sequence having from 4 to 23 contiguousamino acids of the reference sequence, PEPTIDE 1; (b) a sequencesubstantially similar to the amino acid sequence defined in (a); and (c)a variant of the amino acid sequence defined in (a), which variant isselected from the group consisting of a substitution variant, a deletionvariant, an addition variant, and combinations thereof. In someembodiments, the peptides have an amino acid sequence selected from thegroup consisting of: (a) an amino acid sequence having from 8 to 14contiguous amino acids of the reference sequence, PEPTIDE 1; (b) anamino acid sequence substantially identical to the sequence defined in(a); and (c) a variant of the amino acid sequence defined in (a), whichvariant is selected from the group consisting of a substitution variant,a deletion variant, an addition variant, and combinations thereof. Inyet other embodiments, the peptides have an amino acid sequence selectedfrom the group consisting of: (a) an amino acid sequence having from 10to 12 contiguous amino acids of the reference sequence, PEPTIDE 1; (b)an amino acid sequence substantially identical to the sequence definedin (a); and (c) a variant of the amino acid sequence defined in (a),which variant is selected from the group consisting of a substitutionvariant, a deletion variant, an addition variant, and combinationsthereof. In further embodiments, the peptides have an amino acidsequence having at least 9, at least 10, from 9 to 14, from 9 to 13,from 10 to 13, from 10 to 14, or the like contiguous amino acids of thereference sequence, PEPTIDE 1; an amino acid sequence substantiallyidentical thereto; or a variant thereof, which variant is selected fromthe group consisting of a substitution variant, a deletion variant, anaddition variant, and combinations thereof. As explained further below,one or more of the amino acids of the peptides (e.g., the N-terminaland/or C-terminal amino acids) may be optionally independentlychemically modified; in some embodiments, one or more amino acids of apeptide will be chemically modified while in other embodiments none ofthe amino acids of the peptide will be chemically modified. In oneaspect, preferred modification can occur at the amine (H₂N—) group ofthe N-terminal amino acid of the peptide or peptide segment (which aminegroup would form a peptide amide bond if present internally within apeptide sequence rather than at the N-terminal position). In anotheraspect, preferred modification can occur at the carboxy (—COOH) group ofthe C-terminal amino acid of the peptide or peptide segment (whichcarboxy group would form a peptide amide bond if present internallywithin a peptide sequence rather than at the C-terminal position). Inanother aspect, preferred modification can occur at both the N-terminalamine (H₂N—) group and at the C-terminal carboxylic (—COOH) group.

In some embodiments, the amino acid sequence of the peptide begins fromthe N-terminal amino acid of the reference sequence PEPTIDE 1. Forexample, the peptides may have an amino acid sequence selected from thegroup consisting of (a) an amino acid sequence having from 4 to 23contiguous amino acids of the reference sequence PEPTIDE 1, wherein theamino acid sequence begins from the N-terminal amino acid of thereference sequence (i.e., PEPTIDE 2, PEPTIDE 4, PEPTIDE 7, PEPTIDE 11,PEPTIDE 16, PEPTIDE 22, PEPTIDE 29, PEPTIDE 37, PEPTIDE 46, PEPTIDE 56,PEPTIDE 67, PEPTIDE 79, PEPTIDE 92, PEPTIDE 106, PEPTIDE 121, PEPTIDE137, PEPTIDE 154, PEPTIDE 172, PEPTIDE 191, or PEPTIDE 211); (b) asequence substantially similar to the amino acid sequence defined in(a); and (c) a variant of the amino acid sequence defined in (a).

In other embodiments, the amino acid sequence of the peptide ends at theC-terminal amino acid of the reference sequence PEPTIDE 1. For example,the peptides may have an amino acid sequence selected from the groupconsisting of (a) an amino acid sequence having from 4 to 23 contiguousamino acids of the reference sequence PEPTIDE 1, wherein the amino acidsequence ends at the C-terminal amino acid of the reference sequence(i.e., PEPTIDE 3, PEPTIDE 6, PEPTIDE 10, PEPTIDE 15, PEPTIDE 21, PEPTIDE28, PEPTIDE 36, PEPTIDE 45, PEPTIDE 55, PEPTIDE 66, PEPTIDE 78, PEPTIDE91, PEPTIDE 105, PEPTIDE 120, PEPTIDE 136, PEPTIDE 153, PEPTIDE 171,PEPTIDE 190, PEPTIDE 210, or PEPTIDE 231); (b) a sequence substantiallysimilar to the amino acid sequence defined in (a); and (c) a variant ofthe amino acid sequence defined in (a).

In other embodiments, the amino acid sequence of the peptide does notbegin at the N-terminal amino acid of the reference sequence PEPTIDE 1but rather begins at the amino acid at position 2 through the amino acidat position 21 of the reference sequence PEPTIDE 1. For example, thepeptides may have an amino acid sequence selected from the groupconsisting of (a) an amino acid sequence having from 4 to 23 contiguousamino acids of the reference sequence PEPTIDE 1, wherein the amino acidsequence begins at any amino acid between position 2 through position 21of the reference sequence. These peptides may be between 4 and 23contiguous amino acids long and may represent peptides in the middle ofthe reference sequence (PEPTIDE 1; (b) a sequence substantially similarto the amino acid sequence defined in (a); and (c) a variant of theamino acid sequence defined in (a). These peptides are disclose in TableIX.

Peptide amino acid sequences which are useful in the current inventionto inhibit mucin hypersecretion in a mammal, and which are useful toreduce the amount of mucin hypersecretion in a mammal, and which areuseful in the methods of inhibition of mucin hypersecretion and in themethods of reduction of mucin hypersecretion include amino acidsequences of isolated peptides and amino acid sequences of peptideswhich optionally contain N-terminal- and/or C-terminal-chemicallymodified groups of the current invention, which peptide amino acidsequences are selected from the group consisting of the 23-mers (i.e.,peptides having a 23 amino acid sequence): PEPTIDE 2; and PEPTIDE 3; the22-mers (i.e., peptides having a 22 amino acid sequence): PEPTIDE 4;PEPTIDE 5; and PEPTIDE 6; the 21-mers (i.e., peptides having a 21 aminoacid sequence): PEPTIDE 7; PEPTIDE 8; PEPTIDE 9; and PEPTIDE 10; the20-mers (i.e., peptides having a 20 amino acid sequence): PEPTIDE 11;PEPTIDE 12; PEPTIDE 13; PEPTIDE 14; and PEPTIDE 15; the 19-mers (i.e.,peptides having a 19 amino acid sequence): PEPTIDE 16; PEPTIDE 17;PEPTIDE 18; PEPTIDE 19; PEPTIDE 20; and PEPTIDE 21; the 18-mers (i.e.,peptides having a 18 amino acid sequence): PEPTIDE 22; PEPTIDE 23;peptide 25; peptide 26; peptide 27; and peptide 28; the 17-mers (i.e.,peptides having a 17 amino acid sequence): peptide 29; peptide 30;peptide 31; peptide 32; peptide 33; peptide 34; peptide 35; and peptide36; the 16-mers (i.e., peptides having a 16 amino acid sequence):peptide 37; peptide 38; peptide 39; peptide 40; peptide 41; peptide 42;peptide 43; peptide 44; and peptide 45; the 15-mers (i.e., peptideshaving a 15 amino acid sequence): peptide 46; peptide 47; peptide 48;peptide 49; peptide 50; peptide 51; peptide 52; peptide 53; peptide 54;and peptide 55; the 14-mers (i.e., peptides having a 14 amino acidsequence): peptide 56; peptide 57; peptide 58; peptide 59; peptide 60;peptide 61; peptide 62; peptide 63; peptide 64; peptide 65; and peptide66; the 13-mers (i.e., peptides having a 13 amino acid sequence):peptide 67; peptide 68; peptide 69; peptide 70; peptide 71; peptide 72;peptide 73; peptide 74; peptide 75; peptide 76; peptide 77; and peptide78; the 12-mers (i.e., peptides having a 12 amino acid sequence):peptide 79; peptide 80; peptide 81; peptide 82; peptide 83; peptide 84;peptide 85; peptide 86; peptide 87; peptide 88; peptide 89; peptide 90;and peptide 91; the 11-mers (i.e., peptides having a 11 amino acidsequence): peptide 92; peptide 93; peptide 94; peptide 95; peptide 96;peptide 97; peptide 98; peptide 99; peptide 100; peptide 101; peptide102; peptide 103; peptide 104; and peptide 105; the 10-mers (i.e.,peptides having a 10 amino acid sequence): peptide 106; peptide 107;peptide 108; peptide 109; peptide 110; peptide 111; peptide 112; peptide113; peptide 114; peptide 115; peptide 116; peptide 117; peptide 118;peptide 119; and peptide 120; the 9-mers (i.e., peptides having a 9amino acid sequence): peptide 121; peptide 122; peptide 123; peptide124; peptide 125; peptide 126; peptide 127; peptide 128; peptide 129;peptide 130; peptide 131; peptide 132; peptide 133; peptide 134; peptide135; and peptide 136; the 8-mers (i.e., peptides having a 8 amino acidsequence): peptide 137; peptide 138; peptide 139; peptide 140; peptide141; peptide 142; peptide 143; peptide 144; peptide 145; peptide 146;peptide 147; peptide 148; peptide 149; peptide 150; peptide 151; peptide152; and peptide 153; the 7-mers (i.e., peptides having a 7 amino acidsequence): peptide 154; peptide 155; peptide 156; peptide 157; peptide158; peptide 159; peptide 160; peptide 161; peptide 162; peptide 163;peptide 164; peptide 165; peptide 166; peptide 167; peptide 168; peptide169; peptide 170; and peptide 171; the 6-mers (i.e., peptides having a 6amino acid sequence): peptide 172; peptide 173; peptide 174; peptide175; peptide 176; peptide 177; peptide 178; peptide 179; peptide 180;peptide 181; peptide 182; peptide 183; peptide 184; peptide 185; peptide186; peptide 187; peptide 188; peptide 189; and peptide 190; the 5-mers(i.e., peptides having a 5 amino acid sequence): peptide 191; peptide192; peptide 193; peptide 194; peptide 195; peptide 196; peptide 197;peptide 198; peptide 199; peptide 200; peptide 201; peptide 202; peptide203; peptide 204; peptide 205; peptide 206; peptide 207; peptide 208;peptide 209; and peptide 210; and the 4-mers (i.e., peptides having a 4amino acid sequence): peptide 211; peptide 212; peptide 213; peptide214; peptide 215; peptide 216; peptide 217; peptide 218; peptide 219;peptide 220; peptide 221; peptide 222; peptide 223; peptide 224; peptide225; peptide 226; peptide 227; peptide 228; peptide 229; peptide 230;and peptide 231.

Preferred amino acid sequences of isolated peptides and of N-terminal-and/or C-terminal-chemically modified peptides of the current inventionare selected from the group consisting of the 23-mens: PEPTIDE 2; andPEPTIDE 3; the 22-mers: PEPTIDE 4; PEPTIDE 5; and PEPTIDE 6; the21-mers: PEPTIDE 7; PEPTIDE 8; PEPTIDE 9; and PEPTIDE 10; the 20-mers:PEPTIDE 11; PEPTIDE 12; PEPTIDE 13; PEPTIDE 14; and PEPTIDE 15; the19-mers: PEPTIDE 16; PEPTIDE 17; PEPTIDE 18; PEPTIDE 19; PEPTIDE 20; andPEPTIDE 21; the 18-mers: PEPTIDE 22; PEPTIDE 23; peptide 24; peptide 25;peptide 26; peptide 27; and peptide 28; the 17-mers: peptide 29; peptide30; peptide 31; peptide 32; peptide 33; peptide 34; peptide 35; andpeptide 36; the 16-mers: peptide 37; peptide 38; peptide 39; peptide 40;peptide 41; peptide 42; peptide 43; peptide 44; and peptide 45; the15-mers: peptide 46; peptide 47; peptide 48; peptide 49; peptide 50;peptide 51; peptide 52; peptide 53; and peptide 54; the 14-mers: peptide56; peptide 57; peptide 58; peptide 59; peptide 60; peptide 61; peptide62; peptide 63; and peptide 64; the 13-mers: peptide 67; peptide 68;peptide 69; peptide 70; peptide 71; peptide 72; peptide 73; peptide 74;and peptide 75; the 12-mers: peptide 79; peptide 80; peptide 81; peptide82; peptide 83; peptide 84; peptide 85; peptide 86; and peptide 87; the11-mers: peptide 92; peptide 93; peptide 94; peptide 95; peptide 96;peptide 97; peptide 98; peptide 99; and peptide 100; the 10-mers:peptide 106; peptide 107; peptide 108; peptide 109; peptide 110; peptide111; peptide 112; peptide 113; and peptide 114; the 9-mers: peptide 122;peptide 123; peptide 124; peptide 125; peptide 126; peptide 127; peptide128; and peptide 129; the 8-mers: peptide 139; peptide 140; peptide 141;peptide 142; peptide 143; peptide 144; and peptide 145; the 7-mers:peptide 157; peptide 158; peptide 159; peptide 160; peptide 161; andpeptide 162; the 6-mers: peptide 176; peptide 177; peptide 178; peptide179; and peptide 180; the 5-mers: peptide 196; peptide 197; peptide 198;and peptide 199; and the 4-mers: peptide 217; and peptide 219.

More preferred amino acid sequences of isolated peptides and ofN-terminal- and/or C-terminal-chemically modified peptides of thecurrent invention are selected from the group consisting of the 23-mers:peptide 2; and peptide 3; the 22-mers: peptide 4; peptide 5; and peptide6; the 21-mers: peptide 7; peptide 8; peptide 9; and peptide 10; the20-mers: peptide 11; peptide 12; peptide 13; peptide 14; and peptide 15;the 19-mers: peptide 16; peptide 17; peptide 18; peptide 19; peptide 20;and peptide 21; the 18-mers: peptide 22; peptide 23; peptide 24; peptide25; peptide 26; peptide 27; and peptide 28; the 17-mers: peptide 29;peptide 30; peptide 31; peptide 32; peptide 33; peptide 34; peptide 35;and peptide 36; the 16-mers: peptide 37; peptide 38; peptide 39; peptide40; peptide 41; peptide 42; peptide 43; peptide 44; and peptide 45; the15-mers: peptide 46; peptide 47; peptide 48; peptide 49; peptide 50;peptide 51; peptide 52; peptide 53; and peptide 54; the 14-mers: peptide56; peptide 57; peptide 58; peptide 59; peptide 60; peptide 61; peptide62; peptide 63; and peptide 64; the 13-mers: peptide 67; peptide 68;peptide 69; peptide 70; peptide 71; peptide 72; peptide 73; peptide 74;peptide 80; peptide 81; peptide 82; peptide 83; peptide 84; peptide 85;peptide 86; and peptide 87; the 11-mers: peptide 92; peptide 93; peptide94; peptide 95; peptide 96; peptide 97; peptide 98; peptide 99; andpeptide 100; the 10-mers: peptide 106; peptide 108; peptide 109; peptide110; peptide 111; peptide 112; peptide 113; and peptide 114; the 9-mers:peptide 124; peptide 125; peptide 126; peptide 127; peptide 128; andpeptide 129; the 8-mers: peptide 141; peptide 142; peptide 143; peptide144; and peptide 145; the 7-mers: peptide 159; peptide 160; peptide 161;and peptide 162; the 6-mers: peptide 178; peptide 179; and peptide 180;the 5-mers: peptide 198; and peptide 199; and the 4-mer: peptide 219.

In yet other embodiments, the amino acid sequence of the peptideincludes the contiguous residues A,K,G, and E as in peptide 219 of thereference sequence PEPTIDE 1. For example, the peptides may have anamino acid sequence selected from the group consisting of (a) an aminoacid sequence having from 4 to 23 contiguous amino acids of thereference sequence PEPTIDE 1, wherein the amino acid sequence of thepeptide includes the contiguous residues A,K,G, and E as in peptide 219of the reference peptide PEPTIDE 1 (e.g., PEPTIDE 219, PEPTIDE 45,PEPTIDE 79, PEPTIDE 67, PEPTIDE 80, etc.); (b) a sequence substantiallysimilar to the amino acid sequence defined in (a); and (c) a variant ofthe amino acid sequence defined in (a).

Examples of peptide segments which contain the amino acid sequence AKGEof the reference peptide amino acid sequence, PEPTIDE 1, include (a) the23-mers: peptide 2; and peptide 3; the 22-mers: peptide 4; peptide 5;and peptide 6; the 11-mers: peptide 7; peptide 8; peptide 9; and peptide10; the 20-mers: peptide 11; peptide 12; peptide 13; peptide 14; andpeptide 15; the 19-mers: peptide 16; peptide 17; peptide 18; peptide 19;peptide 20; and peptide 21; the 18-mers: peptide 22; peptide 23; peptide24; peptide 25; peptide 26; peptide 27; and peptide 28; the 17-mers:peptide 29; peptide 30; peptide 31; peptide 32; peptide 33; peptide 34;peptide 35; and peptide 36; the 16-mers: peptide 37; peptide 38; peptide39; peptide 40; peptide 41; peptide 42; peptide 43; peptide 44; andpeptide 45; the 15-mers: peptide 46; peptide 47; peptide 48; peptide 49;peptide 50; peptide 51; peptide 52; peptide 53; and peptide 54; the14-mers: peptide 56; peptide 57; peptide 58; peptide 59; peptide 60;peptide 61; peptide 62; peptide 63; and peptide 64; the 13-mers: peptide67; peptide 68; peptide 69; peptide 70; peptide 71; peptide 72; peptide73; peptide 74; and peptide 75; the 12-mers: peptide 79; peptide 80;peptide 81; peptide 82; peptide 83; peptide 84; peptide 85; peptide 86;and peptide 87; the 1′-mers: peptide 93; peptide 94; peptide 95; peptide96; peptide 97; peptide 98; peptide 99; and peptide 100; the 10-mers:peptide 108; peptide 109; peptide 110; peptide 111; peptide 112; peptide113; and peptide 114; the 9-mers: peptide 124; peptide 125; peptide 126;peptide 127; peptide 128; and peptide 129; the 8-mers: peptide 141;peptide 142; peptide 143; peptide 144; and peptide 145; the 7-mers:peptide 159; peptide 160; peptide 161; and peptide 162; the 6-mers:peptide 178; peptide 179; and peptide 180; the 5-mers: peptide 198; andpeptide 199; and the 4-mer: peptide 219, (b) a sequence substantiallysimilar to the amino acid sequence defined in (a); and (c) a variant ofthe amino acid sequence defined in (a), which variant is selected fromthe group consisting of a substitution variant, a deletion variant, anaddition variant, and combinations thereof, wherein the segmentcomprises or consists of from 4 to 23 contiguous amino acids.

In another embodiment, preferred peptide sequences have an amino acidsequence selected from the group consisting of (a) an amino acidsequence having from 10 to 23 contiguous amino acids of the referencesequence, peptide 1; (b) a sequence substantially similar to the aminoacid sequence defined in (a); and (c) a variant of the amino acidsequence defined in (a), which variant is selected from the groupconsisting of a substitution variant, a deletion variant, an additionvariant, and combinations thereof, wherein the preferred amino acidsequences comprise the 23-mer: peptide 2; the 22-mer: peptide 4; the21-mer: peptide 7; the 20-mer: peptide 11; the 19-mer: peptide 16; the18-mer: peptide 22; the 17-mer: peptide 29; the 16-mer: peptide 37; the15-mer: peptide 46; the 14-mer: peptide 56; the 13-mer: peptide 67; the12-mer: peptide 79; the 11-mer: peptide 92; and the 10-mer: peptide 106.

In further embodiments, the amino acid sequence of the peptide beginsfrom the N-terminal amino acid of the reference sequence PEPTIDE 1 andincludes the contiguous residues A,K,G, and E as in peptide 219 of thereference sequence PEPTIDE 1, while in other embodiments the amino acidsequence of the peptide ends at the C-terminal amino acid of thereference sequence PEPTIDE 1 and includes the contiguous residues A,K,G,and E as in peptide 219 of the reference sequence PEPTIDE 1.

The peptides may include one or more amino acid deletions,substitutions, and/or additions with respect to the reference amino acidsequence. Preferably, the substitutions may be conservative amino acidsubstitutions, or the substitutions may be non-conservative amino acidsubstitutions. In some embodiments, the peptides, including the peptideswith amino acid sequences that are substantially identical to orvariants of the reference amino acid sequence, will not have deletionsor additions as compared to the corresponding contiguous amino acids ofthe reference amino acid sequence, but may have conservative ornon-conservative substitutions. Amino acid substitutions that may bemade to the reference amino acid sequence in the peptides of theinvention include, but are not limited to, the following: alanine (A)may be substituted with lysine (K), valine (V), leucine (L), orisoleucine (I); glutamic acid (E) may be substituted with aspartic acid(D); glycine (G) may be substituted with proline (P); lysine (K) may besubstituted with arginine (R), glutamine (Q), or asparagine (N);phenylalanine (F) may be substituted with leucine (L), valine (V),isoleucine (I), or alanine (A); proline (P) may be substituted withglycine (G); glutamine (Q) may be substituted with glutamic acid (E) orasparagine (N); arginine (R) may be substituted with lysine (K),glutamine (Q), or asparagine (N); serine (S) may be substituted withthreonine; threonine (T) may be substituted with serine (S); and valine(V) may be substituted with leucine (L), isoleucine (I), methionine (M),phenylalanine (F), alanine (A), or norleucine (Nle). For example,substitutions that could be made to the reference amino acid sequence inthe peptides of the invention include substituting alanine (A) forphenylalanine (F) (e.g., at amino acid position 4 of the reference aminoacid sequence), glutamic acid (E) for glutamine (Q) (e.g., at amino acidposition 3 of the reference amino acid sequence), lysine (K) for alanine(A) (e.g., at amino acid positions 2 and/or 8 of the reference aminoacid sequence), and/or serine (S) for threonine (T) (e.g., at amino acidposition 7 of the reference amino acid sequence).

When substitutions are included in the amino acid sequences of thepeptides of the invention (which peptides comprise unmodified as well aspeptides which are chemically modified for example by N-terminal and/orC-terminal modification such as by amide formation) with respect to thereference amino acid sequence, there is preferably at least 80% sequenceidentity between the amino acid sequence of the peptide and thereference amino acid sequence. Peptides having 5 to 23 amino acids andincluding one amino acid substitution with respect to the referenceamino acid sequence will have between about 80% to about 96% (i.e.,˜95.7%) sequence identity to the reference amino acid sequence. Peptideshaving 10 to 23 amino acids and including one amino acid substitutionwith respect to the reference amino acid sequence will have betweenabout 90% to about 96% (i.e., ˜95.7%) sequence identity to the referenceamino acid sequence. Peptides having 20 to 23 amino acids and includingone amino acid substitution with respect to the reference amino acidsequence will have between about 95% to about 96% (i.e., ˜95.7%)sequence identity to the reference amino acid sequence. Peptides having10 to 23 amino acids and including two amino acid substitutions withrespect to the reference amino acid sequence will have between about 80%to about 92% (i.e., ˜91.3%) sequence identity to the reference aminoacid sequence. Peptides having 16 to 23 amino acids and including twoamino acid substitutions with respect to the reference amino acidsequence will have between about 87.5% to about 92% (i.e., ˜91.3%)sequence identity to the reference amino acid sequence. Peptides having20 to 23 amino acids and including two amino acid substitutions withrespect to the reference amino acid sequence will have between about 90%to about 92% (i.e., ˜91.3%) sequence identity to the reference aminoacid sequence. Peptides having 15 to 23 amino acids and including threeamino acid substitutions with respect to the reference amino acidsequence will have between about 80% to about 87% sequence identity tothe reference amino acid sequence. Peptides having 20 to 23 amino acidsand including three amino acid substitutions with respect to thereference amino acid sequence will have between about 85% to about 87%sequence identity to the reference amino acid sequence. Peptides having20 to 23 amino acids and including four amino acid substitutions withrespect to the reference amino acid sequence will have between about 80%to about 83% (i.e., ˜82.6%) sequence identity to the reference aminoacid sequence.

In peptides of the current invention, with respect to the contiguousamino acid sequence of the reference peptide (which is a 24-mer)substitution of one amino acid in a contiguous 23 amino acid sequence (a23-mer) selected from the reference 24 amino acid sequence provides apeptide with an amino acid sequence which has a 95.65% (or ˜96%)sequence identity to the amino acid segment in the reference peptidewith which the 23-mer has identity. Analogously, substitution of two,three, four, and five amino acids in said 23-mer provides a peptide withan amino acid sequence which has a 91.30% (or ˜91%), 86.96% (or ˜87%),82.61% (or ˜83%), and 78.27% (or ˜78%) sequence identity, respectively,to the reference peptide amino acid sequence. Analogously, substitutionof one, two, three, four, and five amino acids in a 22-mer provides apeptide with an amino acid sequence which has a 95.45% (or ˜95%), 90.91%(or ˜91%), 86.36% (or ˜86%), 81.82% (or ˜82%), and 77.27% (or ˜77%)sequence identity, respectively, to the reference peptide amino acidsequence Analogously, substitution of one, two, three, four, and fiveamino acids in a 21-mer provides a peptide with an amino acid sequencewhich has a 95.24% (˜95%), 90.48 (˜91%), 85.71% (˜86%), 80.95 (˜81%),and 76.19% (˜76%) sequence identity, respectively, to the referencepeptide amino acid sequence. Analogously, substitution of one, two,three, four, and five amino acids in a 20-mer provides a peptide with anamino acid sequence which has a 95.00% (95%), 90.00% (90%), 85.00%(85%), 80.00% (80%), and 75.00% (75%) sequence identity, respectively,to the reference peptide amino acid sequence. Analogously, substitutionof one, two, three, and four amino acids in a 19-mer provides a peptidewith an amino acid sequence which has a 94.74% (˜95%), 89.47% (˜89%),84.21% (˜84%), and 78.95% (˜79%) sequence identity, respectively, to thereference peptide amino acid sequence. Analogously, substitution of one,two, three, and four amino acids in an 18-mer provides a peptide with anamino acid sequence which has a 94.44% (˜94%), 88.89% (˜89%), 83.33%(˜83%), and 77.78% (˜78%) sequence identity, respectively, to thereference peptide amino acid sequence. Analogously, substitution of one,two, three, and four amino acids in an 17-mer provides a peptide with anamino acid sequence which has a 94.12% (˜94%), 88.23% (˜88%), 82.35%(˜82%), and 76.47% (˜76%) sequence identity, respectively, to thereference peptide amino acid sequence. Analogously, substitution of one,two, three, and four amino acids in a 16-mer provides a peptide with anamino acid sequence which has a 93.75% (˜94%), 87.50% (˜88%), 81.25%(˜81%), and 75.00% (75%) sequence identity, respectively, to thereference peptide amino acid sequence. Analogously, substitution of one,two, and three amino acids in a 15-mer provides a peptide with an aminoacid sequence which has a 93.33% (˜93%), 86.67% (˜87%), and 80.00% (80%)sequence identity, respectively, to the reference peptide amino acidsequence. Analogously, substitution of one, two, and three amino acidsin a 14-mer provides a peptide with an amino acid sequence which has a92.86% (˜93%), 85.71% (˜86%), and 78.57% (79%) sequence identity,respectively, to the reference peptide amino acid sequence. Analogously,substitution of one, two, and three amino acids in a 13-mer provides apeptide with an amino acid sequence which has a 92.31% (˜92%), 84.62%(˜85%), and 76.92% (˜77%) sequence identity, respectively, to thereference peptide amino acid sequence. Analogously, substitution of one,two, and three amino acids in a 12-mer provides a peptide with an aminoacid sequence which has a 91.67% (˜92%), 83.33% (˜83%), and 75.00% (75%)sequence identity, respectively, to the reference peptide amino acidsequence. Analogously, substitution of one and two amino acids in an11-mer provides a peptide with an amino acid sequence which has a 90.91%(˜91%) and 81.82% (˜82%) sequence identity, respectively, to thereference peptide amino acid sequence. Analogously, substitution of oneand two amino acids in a 10-mer provides a peptide with an amino acidsequence which has a 90.00% (90%) and 80.00% (80%) sequence identity,respectively, to the reference peptide amino acid sequence. Analogously,substitution of one and two amino acids in a 9-mer provides a peptidewith an amino acid sequence which has a 88.89% (˜89%) and 77.78% (˜78%)sequence identity, respectively, to the reference peptide amino acidsequence. Analogously, substitution of one and two amino acids in an8-mer provides a peptide with an amino acid sequence which has a 87.50%(˜88%) and 75.00% (75%) sequence identity, respectively, to thereference peptide amino acid sequence. Analogously, substitution of oneamino acid in a 7-mer, 6-mer, 5-mer, and 4-mer provides a peptide withan amino acid sequence which has a 85.71% (˜86%), 83.33% (˜83.3%),80.00% (80%), and 75.00% (75%) sequence identity, respectively, to thereference peptide. Preferred amino acid sequences of this invention havegreater than 80% sequence identity to the amino acid sequence in thereference sequence, more preferably between 81% and 96% sequenceidentity to the amino acid sequence in the reference sequence, and morepreferably between 80% and 96% sequence identity to the amino acidsequence in the reference sequence. The preferred amino acid sequencescan be optionally N-terminally chemically bonded at the terminal peptideamino group to a C₂ to C₂₂ linear aliphatic carboxylic acid moiety, morepreferably to a C₂ to C₁₆ linear aliphatic carboxylic acid moiety, mostpreferably to a C₂ or C₁₆ linear aliphatic carboxylic acid moiety, by anamide bond, and optionally C-terminally chemically bonded at theterminal peptide carboxylic group to an amine such as ammonia or aprimary or secondary amine such as a C1 to C16 linear aliphatic primaryamine, by an amide bond.

Examples of substitution variants of peptide 79, a 12-mer, include, forexample, peptide 238, where Q at position 3 in peptide 79 has beensubstituted by E in sequence 238; peptide 233, where A at position 2 inpeptide 79 has been substituted by K in peptide 233; peptide 234, whereA at position 8 in peptide 79 has been substituted by K in peptide 234;peptide 235, where A at positions 2 and 8 in peptide 79 have beensubstituted by K in peptide 235; peptide 237, where F at position 4 inpeptide 79 has been substituted by A in peptide 237; peptide 239, whereK at position 10 in peptide 79 has been substituted by A in peptide 239;peptide 240, where G at position 11 in peptide 79 has been substitutedby A in peptide 240; and peptide 241, where F at position 12 in peptide79 has been substituted by A in peptide 241.

Examples of substitution variants of peptide 106, a 10-mer, include, forexample, peptide 236, where F at position 4 in peptide 106 has beensubstituted by A in peptide 236; peptide 242, where G at position 1 inpeptide 106 has been substituted by A in peptide 242; peptide 243, whereQ at position 3 in peptide 106 has been substituted by A in peptide 243;peptide 244, where S at position 5 in peptide 106 has been substitutedby A in peptide 244; peptide 245, where K at position 6 in peptide 106has been substituted by A in peptide 245; peptide 247, where T atposition 7 in peptide 106 has been substituted by A in peptide 247;peptide 248, where K at position 10 in peptide 106 has been substitutedby A in peptide 248; peptide 249, where K at positions 6 and 10 inpeptide 106 have both been substituted, each by A, in peptide 249.

Examples of a substitution variant of peptide 137, an 8-mer, include forexample, peptide 250, where F at position 4 in peptide 137 has beensubstituted by A in peptide 250.

Examples of a substitution variant of peptide 219, a 4-mer, include forexample, peptide 251, where K at position 2 in peptide 219 has beensubstituted by A in peptide 251.

A substitution variant peptide such as described herein can be in theform of an isolated peptide or in the form of a chemically modifiedpeptide such as, for example, an N-terminal amide such as a myristoylamide, an acetyl amide, and the like as described herein, and such as,for example, a C-terminal amide such as an amide formed with ammonia,and such as both an N-terminal amide and a C-terminal amide.

When deletions are included in the amino acid sequences of the peptidesof the invention with respect to the reference amino acid sequence,there is preferably at least 80% sequence identity between the aminoacid sequence of the peptide to the reference amino acid sequence.Peptides having 5 to 23 amino acids and including one amino aciddeletion with respect to the reference peptide will have between 80% toabout 96% (i.e., ˜95.7%) sequence identity to the reference amino acidsequence. Peptides having 10 to 23 amino acids and including one aminoacid deletion with respect to the reference peptide will have betweenabout 90% to about 96% (i.e., ˜95.7%) sequence identity to the referenceamino acid sequence. Peptides having 20 to 23 amino acids and includingone amino acid deletion with respect to the reference peptide will havebetween 95% to about 96% (i.e., ˜95.7%) sequence identity to thereference amino acid sequence. Peptides having 10 to 23 amino acids andincluding two amino acid deletions with respect to the reference peptidewill have between about 80% to about 92% (i.e., ˜91.3%) sequenceidentity to the reference amino acid sequence. Peptides having 16 to 23amino acids and including two amino acid deletions with respect to thereference peptide will have between about 87.5% to about 92% (i.e.,˜91.3%) sequence identity to the reference amino acid sequence. Peptideshaving 20 to 23 amino acids and including two amino acid deletions withrespect to the reference peptide will have between about 90% to about92% (i.e., ˜91.3%) sequence identity to the reference amino acidsequence. Peptides having 15 to 23 amino acids and including three aminoacid deletions with respect to the reference peptide will have betweenabout 80% to about 87% sequence identity to the reference amino acidsequence. Peptides having 20 to 23 amino acids and including three aminoacid deletions with respect to the reference peptide will have betweenabout 85% to about 87% sequence identity to the reference amino acidsequence. Peptides having 20 to 23 amino and including four amino aciddeletions with respect to the reference peptide will have between about80% to about 83% (i.e., ˜82.6%) sequence identity to the reference aminoacid sequence.

As stated above, one or more of the amino acids of the peptides may alsobe chemically modified. Any amino acid modifications known in the artmay be made to the amino acids of the peptides using any method known inthe art.

In some embodiments, the N-terminal and/or C-terminal amino acid may bemodified. For example, the N-terminal amino acid of the peptides may bealkylated, amidated, or acylated at the N-terminal amino (H₂N—) group,and, for example, the C-terminal amino acid of the peptides may beamidated or esterified at the C-terminal carboxyl (—COOH) group. Forexample, the N-terminal amino group may be modified by acylation toinclude any acyl or fatty acyl group to form an amide, including anacetyl group (i.e., CH₃—C(═O)— or a myristoyl group. In someembodiments, the N-terminal amino group may be modified to include anacyl group having formula —C(O)R, wherein R is a linear or branchedalkyl group having from 1 to 15 carbon atoms, or may be modified toinclude an acyl group having formula —C(O)R¹, wherein R¹ is a linearalkyl group having from 1 to 15 carbon atoms. The C-terminal amino acidof the peptides may also be chemically modified. For example, theC-terminal carboxyl group of the C-terminal amino acid may be chemicallymodified to include an amino group in place of the hydroxyl group.(i.e., amidated). In some embodiments, the N-terminal and/or C-terminalamino acids are not chemically modified.

The peptide may be acylated at the amino group of the N-terminal aminoacid to form an N-terminal amide with an acid selected from the groupconsisting of:

(i) a C₂ to C₂₄ aliphatic (saturated or optionally unsaturated)carboxylic acid (for example, an N-terminal amide with acetic acid, withpropanoic acid, with butanoic acid, with hexanoic acid, with octanoicacid, with decanoic acid, with dodecanoic acid, with tetradecanoic acid(myristic acid), with hexadecanoic acid, with 9-hexadecenoic acid, withoctadecanoic acid, with 9-octadecenoic acid, with 11-octadecenoic acid,with 9,12-octadecadienoic acid, with 9,12,15-octadecatrienoic acid, with6,9,12-octadecatrienoic acid, with eicosanoic acid, with 9-eicosenoicacid, with 5,8,11,14-eicosatetraenoic acid, with5,8,11,14,17-eicosapentaenoic acid, with docosanoic acid, with13-docosenoic acid, with 4,7,10,13,16,19-docosahexaenoic acid, withtetracosanoic acid, and the like);

(ii) trifluoroacetic acid;

(iii) benzoic acid; and

(iv) a C₁ to C₂₄ aliphatic alkyl sulfonic acid which forms an aliphaticalkyl sulfonamide, wherein the C₁ to C₂₄ aliphatic alkyl carbon chainstructure of the sulfonic acid is analogous to that of the aliphaticalkyl carboxylic acid chains in the aliphatic alkyl carboxylic acidsdescribed above. For example, a peptide may be acylated using acarboxylic acid group represented as (C₁-C₂₃)-alkyl-C(O)OH throughdehydrative coupling by way of activation of the carboxylic acid groupto form an amide represented as (C₁-C₂₃)-alkyl-C(O)—NH-peptide.Analogously, a sulfonamide may be formed by reacting a sulfonic acidspecies (represented as (C₁-C₂₃)-alkyl-S(O₂)—X, where X is halogen orOCH₃ or other compatible leaving group) with an N-terminal amino groupto form a sulfonamide represented as (C₁-C₂₃)-alkyl-S(O₂)—NH-peptide.

As another example, the N-terminal amino group of the N-terminal aminoacid may be alkylated with a C₁ to C₂₄ aliphatic alkyl group, thestructure of which aliphatic alkyl group is as described above.Alkylation may be effected, for example, using an aliphatic alkyl halideor an aliphatic alkyl sulfonic acid ester (mesylate, tosylate, etc.),preferably using a primary alkyl halide or a primary alkyl sulfonic acidester. The N-terminal amino acid may be also modified at the terminalamino to include any acyl or aliphatic acyl fatty acyl group as anamide, including an acetyl group (i.e., —C(O)CH₃), a myristoyl group, abutanoyl group, a hexanoyl group, a octanoyl group, a decanoyl group, adodecanoyl group, a tetradecanoyl group, a hexadecanoyl group, a9-hexadecenoyl group, a octadecanoyl group, a 9-octadecenoyl group, a11-octadecenoyl group, a 9,12-octadecadienoyl group, a9,12,15-octadecatrienoyl group, a 6,9,12-octadecatrienoyl group, aeicosanoyl group, a 9-eicosenoyl group, a 5,8,11,14-eicosatetraenoylgroup, a 5,8,11,14,17-eicosapentaenoyl group, a docosanoyl group, a13-docosenoyl group, a 4,7,10,13,16,19-docosahexaenoyl group, atetracosanoyl group, which groups are covalently attached to theterminal amino group of the peptide by an amide bond.

The C-terminal carboxylic acid group of the C-terminal amino acid of thepeptides of the invention may also be chemically modified. For example,the C-terminal amino acid may be chemically modified by reaction of theC-terminal carboxylic acid group of the peptide with an amine to form anamide group such as an amide of ammonia; an amide of a C₁ to C₂₄aliphatic alkyl amine, preferably a linear aliphatic alkyl amine; anamide of a hydroxyl-substituted C₂ to C₂₄ aliphatic alkyl amine; anamide of a linear 2-(C₁ to C₂₄ aliphatic alkyl)oxyethylamine group; andan amide of an omega-methoxy-poly(ethyleneoxy)_(n)-ethylamine group(also referred to as an omega-methoxy-PEG-alpha-amine group or anomega-methoxy-(polyethylene glycol)amine group), where n is from 0 to10. The C-terminal carboxylic acid group of the C-terminal amino acid ofthe peptide may also be in the form of an ester selected from the groupconsisting of an ester of a C₁ to C₂₄ aliphatic alkyl alcohol and anester of a 2-(omega-methoxy-poly(ethyleneoxy)_(n))-ethanol group, wheren is from 0 to 10.

The C-terminal carboxylic acid group on the peptide, which may berepresented by the formula peptide-C(O)OH, may also be amidated byconversion to an activated form such as a carboxylic acid halide,carboxylic acid anhydride, N-hydroxysuccinimide ester, pentafluorophenyl(OPfp) ester, 3-hydroxy-2,3-dihydro-4-oxo-benzo-triazone (ODhbt) ester,and the like to facilitate reaction with ammonia or a primary orsecondary amine, preferably ammonia or a primary amine, and preferablywhile any other reactive groups in the peptide are protected bysynthetic chemically compatible protecting groups well known in the artof peptide synthesis, especially of peptide solid phase synthesis, suchas a benzyl ester, a t-butyl ester, a phenyl ester, etc. A resultingpeptide amide could be represented by the formula peptide-C(O)—NR³R⁴(the amide being at the C-terminal end of the peptide) wherein R³ and R⁴are independently selected from the group consisting of hydrogen; C₁ toC₂₄ alkyl such as methyl, ethyl, butyl, isobutyl, cyclopropylmethyl,hexyl, dodecyl, tetradecyl, and the like as described above.

The C-terminal carboxylic acid of the C-terminal amino acid may also beconverted to an amide of a hydroxyl-substituted C₂ to C₂₄ aliphaticalkyl amine (the hydroxyl group being attached to a carbon atom ratherthan a nitrogen atom of the amine) such as 2-hydroxyethylamine,4-hydroxybutylamine, and 12-hydroxydodecylamine, and the like.

The C-terminal carboxylic acid may also be converted to an amide of ahydroxyl-substituted C₂ to C₂₄ aliphatic alkyl amine, wherein thehydroxyl group could be acylated to form an ester with a C₂ to C₂₄aliphatic carboxylic acid as described above. Preferably, in the peptideamide at the C-terminal end of the peptide represented by the formulapeptide-C(O)R⁵R⁶, R⁵ is hydrogen and R⁶ is selected from the groupconsisting of hydrogen, C₁ to C₂₄ alkyl, and hydroxyl-substituted C₂ toC₂₄ alkyl.

The C-terminal carboxylic acid of the C-terminal amino acid may beconverted to an amide of a linear 2-(C₁ to C₂₄ aliphaticalkyl)oxyethylamine. Such an amide may be prepared, for example, byreaction of a linear C₁ to C₂₄ aliphatic alcohol with potassium hydridein diglyme with 2-chloroethanol to provide a linear C₁ to C₂₄ aliphaticalkyl ethanol, which can be converted to an amine by oxidation to analdehyde, followed by reductive amination to an amine (for example usingammonia), or by conversion to an alkyl halide (e.g. using thionylchloride) followed by treatment with an amine such as ammonia.

The C-terminal carboxylic acid of the C-terminal amino acid may also beconverted to an amide of anomega-methoxy-poly(ethyleneoxy)_(n)-ethylamine, where n is from 0 to 10,which can be prepared from the correspondingomega-methoxy-poly(ethyleneoxy)_(n)-ethanol, for example, by conversionof the alcohol to an amine as described above.

In another embodiment, the C-terminal carboxyl may be converted to anamide represented by the formula peptide-C(O)—NR⁷R⁸, wherein R⁷ ishydrogen and R⁸ is a linear 2-(C₁ to C₂₄ aliphatic alkyl)oxyethyl groupwherein the C₁ to C₂₄ aliphatic alkyl portion is as described above andincludes groups such as methoxyethyl (i.e., CH₃O—CH₂CH₂—),2-dodecyloxyethyl, and the like; or R⁷ is hydrogen and R⁸ is anomega-methoxy-poly(ethyleneoxy)_(n)-ethyl group where the n of thepoly(ethyleneoxy) portion is from 0 to 10, such as 2-methoxyethyl (i.e.,CH₃O—CH₂CH₂—), omega-methoxyethoxyethyl (i.e., CH₃O—CH₂CH₂O—CH₂CH₂—) upto CH₃O—(CH₂CH₂O)₁₀—CH₂CH₂—.

The C-terminal carboxylic acid group of the C-terminal amino acid of thepeptide may also be in the form of an ester of a C₁ to C₂₄ aliphaticalkyl alcohol, the aliphatic alkyl portion of the alcohol as describedabove. The C-terminal carboxylic acid group of the C-terminal amino acidof the peptide may also be in the form of an ester of a2-(omega-methoxy-poly(ethyleneoxy)_(n))-ethanol group where n is from 0to 10, which can be prepared from reaction of 2-methoxyethanol as asodium 2-methoxyethanolate with stoichiometric amounts of ethyleneoxide, the stoichiometric amount dependent on the size of n.

A side chain in an amino acid of the peptides may also be chemicallymodified. For example, a phenyl group in phenylalanine or tyrosine maybe substituted with a substituent selected from the group consisting of:

a C₁ to C₂₄ aliphatic alkyl group (i.e., linear or branched, and/orsaturated or unsaturated, and/or containing a cyclic group) such asmethyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopropyl,2-methylcyclopropyl, cyclohexyl, octyl, decyl, dodecyl, hexadecyl,octadecyl, eicosanyl, docosanyl, tetracosanyl, 9-hexadecenyl,9-octadecenyl, 11-octadecenyl, 9,12-octadecadienyl,9,12,15-octadecatrienyl, 6,9,12-octadecatrienyl, 9-eicosenyl,5,8,11,14-eicosatetraenyl, 5,8,11,14,17-eicosapentaenyl, 13-docosenyl,and 4,7,10,13,16,19-docosahexaenyl;

a C₁ to C₂₄ aliphatic alkyl group substituted with a hydroxyl group atleast one carbon atom away from a site of unsaturation, examples ofwhich hydroxyalkyl group include hydroxymethyl, hydroxyethyl,hydroxydodecyl, and the like;

a C₁ to C₂₄ alkyl group substituted with a hydroxyl group that isesterified with a C₂ to C₂₄ aliphatic carboxyl group of an acid such asacetic acid, butanoic acid, hexanoic acid, octanoic acid, decanoic acid,dodecanoic acid, tetradecanoic acid, hexadecanoic acid, 9-hexadecenoicacid, octadecanoic acid, 9-octadecenoic acid, 11-octadecenoic acid,9,12-octadecadienoic acid, 9,12,15-octadecatrienoic acid,6,9,12-octadecatrienoic acid, eicosanoic acid, 9-eicosenoic acid,5,8,11,14-eicosatetraenoic acid, 5,8,11,14,17-eicosapentaenoic acid,docosanoic acid, 13-docosenoic acid, 4,7,10,13,16,19-docosahexaenoicacid, tetracosanoic acid, and the like, a dicarboxylic acid such assuccinic acid, or a hydroxyacid such as lactic acid, wherein the totalnumber of carbon atoms of the ester substituent is between 3 and 25;

halogen such as fluoro-, chloro-, bromo-, and iodo-; nitro-; amino- suchas NH₂, methyl amino, dimethylamino; trifluoromethyl-; carboxyl (—COOH);

a C₁ to C₂₄ alkoxy (such as can be formed by alkylation of tyrosine)such as methoxy, ethoxy, propyloxy, isopropyloxy, butyloxy, isobutyloxy,cyclopropyloxy, 2-methoxycyclopropyloxy, cyclohexyloxy, octyloxy,decyloxy, dodecyloxy, hexadecyloxy, octadecyloxy, eicosanyloxy,docosanyloxy, tetracosanyloxy, 9-hexadecenyloxy, 9-octadecenyloxy,11-octadecenyloxy, 9,12-octadecadienyloxy, 9,12,15-octadecatrienyloxy,6,9,12-octadecatrienyloxy, 9-eicosenyloxy, 5,8,11,14-eicosatetraenyloxy,5,8,11,14,17-eicosapentaenyloxy, 13-docosenyloxy, and4,7,10,13,16,19-docosahexaenyloxy; and

a C₂ to C₂₄ hydroxyalkyloxy such as 2-hydroxyethyloxy and esters thereofwith carboxylic acids as described above or with trifluoroacetic acid.

A serine hydroxyl group may be esterified with a substituent selectedfrom the group consisting of:

a C₂ to C₂₄ aliphatic carboxylic acid group such as described above;

a trifluoroacetic acid group; and

a benzoic acid group.

The epsilon amino group in lysine may be chemically modified, forexample, by amide formation with: a C₂ to C₂₄ aliphatic carboxylic acidgroup (for example, by reaction of the amine with a chemically activatedform of a carboxylic acid such as an acid chloride, an anhydride, anN-hydroxysuccinimide ester, a pentafluorophenyl (OPfp) ester, a3-hydroxy-2,3-dihydro-4-oxo-benzo-triazone (ODhbt) ester, and the like)such as described above, or a benzoic acid group, or an amino acidgroup. Additionally, the epsilon amino group in lysine may be chemicallymodified by alkylation with one or two C₁ to C₄ aliphatic alkyl groups.

The carboxylic acid group in glutamic acid may be modified by formationof an amide with an amine such as: ammonia; a C₁ to C₂₄ primaryaliphatic alkyl amine (the alkyl portion of which is as described above)including with methylamine; or an amino group of an amino acid.

The carboxylic acid group in glutamic acid may be modified by formationof an ester with a C₁ to C₂₄ aliphatic hydroxyalkyl group as describedabove, preferably an ester with a primary alcohol of a C₁ to C₂₄aliphatic alkyl such as methanol, ethanol, propan-1-ol, n-dodecanol, andthe like as described above.

The peptides of the invention have a mucin-inhibiting effect and/ormucus-inhibiting effect when administered to a mammal in a mucin- and/ormucus-inhibiting amount. The peptides may also have (1) a greatermucin-inhibiting effect in a mammal than MANS peptide when administeredat equal concentrations, (2) a greater mucus-inhibiting effect in amammal than MANS peptide when administered at equal concentrations,and/or (3) have greater aqueous solubility than the MANS peptide.

The MARCKS peptide and the MANS peptide each comprise a myristoyl grouplinked to the amine at the N-terminal amino acid by an amide bond.However, as disclosed herein, mucin hypersecretion-inhibiting activityof the peptides of the invention does not reside with the presence of amyristoyl group at the N-terminal amino acid of the peptide sequence.Indeed, certain peptides of the invention which do not contain anN-terminal chemical modification are found to exhibit mucinhypersecretion-inhibiting activity. Certain peptides of the inventionwhich contain N-terminal chemical modification by groups other thanmyristoyl, such as an acetyl group as an N-terminal amide also are foundto exhibit mucin hypersecretion-inhibiting activity. Indeed, anN-terminal acetylated version of the MANS peptide (non-myristylated) canexhibit mucin hypersecretion-inhibiting activity in the methods of thisinvention. In addition, the peptide sequence of amino acids comprisingthe MANS peptide amino acid sequence and variants thereof as describedherein can exhibit mucin hypersecretion-inhibiting activity in themethods of this invention.

In one aspect, this invention provides a method of inhibiting mucinhypersecretion in a mammal, the method comprising administering to themammal a mucin hypersecretion-inhibiting amount of a peptide thatinhibits mucin hypersecretion, the peptide having an amino acid sequenceselected from the group consisting of: (a) an amino acid sequence havingthe sequence, GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO. 1); and (b) an aminoacid sequence substantially identical to the sequence defined in (a);wherein the N-terminal amino acid of the peptide is not myristoylatedand the C-terminal amino acid of the peptide is optionally independentlychemically modified, the peptide having a mucinhypersecretion-inhibiting effect when administered to a mammal in amucin hypersecretion-inhibiting amount. In one embodiment, theN-terminal amino acid of this peptide is preferably acetylated. Inanother embodiment, the peptide exhibits at least one of the propertiesof (a) greater mucin hypersecretion-inhibiting effect on a mammal thanSEQ ID NO. 1, wherein the N-terminal amino acid is myristoylated whenadministered to said mammal at equal concentrations or (b) greateraqueous solubility than SEQ ID NO. 1, wherein the N-terminal amino acidis myristoylated at equal concentrations in the same liquid.

In some embodiments, the N-terminal amino group may be modified toinclude an acyl group having formula —C(O)R, wherein R is a linear orbranched alkyl group having from 1 to 15 carbon atoms, or may bemodified to include an acyl group having formula —C(O)R¹, wherein R¹ isa linear alkyl group having from 1 to 15 carbon atoms. The C-terminalamino acid of the peptides may also be chemically modified. For example,the C-terminal carboxyl group of the C-terminal amino acid may bechemically modified to include an amino group in place of the hydroxylgroup. (i.e., amidated). In some embodiments, the N-terminal and/orC-terminal amino acids are not chemically modified.

In some embodiments, the peptides may have a greater half-life in humanbronchoalveolar lavage fluid (BALF) than in human plasma, and may alsohave a greater half-life in human mucus (e.g., in mucus from a subjectwith cystic fibrosis) than in human plasma.

The peptides may be used in methods of inhibiting mucin secretion and/ormucus production in a mammal, methods of treating hypersecretion ofmucin and/or mucus in a mammal, and methods of treating pulmonarydiseases causing hypersecretion of mucin and/or mucus in a mammal (suchas, for example, asthma, chronic bronchitis, COPD, and cystic fibrosis).Conditions that may be suitable for treatment by the peptides include,but are not limited to, inflammatory, viral, or bacterial airway disease(e.g., asthma, chronic obstructive pulmonary disease (COPD), commoncold, rhinitis, acute or chronic bronchitis, pneumonia, and kennelcough), allergic conditions (e.g., atopy, allergic inflammation),bronchiectasis, emphysema, bronchial asthma, and certain geneticconditions (e.g., cystic fibrosis). The peptides may also be suitablefor treatment of conditions and diseases described in, as well as formethods described in, U.S. patent application Ser. Nos. 10/180,753(Publication No. U.S. 2003/0013652) and 09/256,154 and InternationalApplication No. PCT/US00/05050 (International Publication Number WO00/50062), the entire contents of which are incorporated herein byreference.

In addition to mucin hypersecretion associated with a disease, the termmucin hypersecretion also includes ATP-induced mucin hypersecretion aswell as secretagogue-induced mucin hypersecretion and stimulated mucinhypersecretion.

In a preferred embodiment, the current invention provides a mucinhypersecretion-inhibiting peptide having an amino acid sequence of from4 to 23 contiguous amino acids of a reference amino acid sequence,PEPTIDE 1, wherein the mucin hypersecretion-inhibiting peptide isselected from:

(a) the group consisting of peptide 2, peptide 3, peptide 4, peptide 5,peptide 6, peptide 7, peptide 8, peptide 9, peptide 10, peptide 11,peptide 12, peptide 13, peptide 14, peptide 15, peptide 16, peptide 17,peptide 18, peptide 19, peptide 20, peptide 21, peptide 22, peptide 23,peptide 24, peptide 25, peptide 26, peptide 27, peptide 28, peptide 29,peptide 30, peptide 31, peptide 32, peptide 33, peptide 34, peptide 35,peptide 36, peptide 37, peptide 38, peptide 39, peptide 40, peptide 41,peptide 42, peptide 43, peptide 44, peptide 45, peptide 46, peptide 47,peptide 48, peptide 49, peptide 50, peptide 51, peptide 52, peptide 53,peptide 54, peptide 56, peptide 57, peptide 58, peptide 59, peptide 60,peptide 61, peptide 62, peptide 63, peptide 64, peptide 67, peptide 68,peptide 69, peptide 70, peptide 71, peptide 72, peptide 73, peptide 74,peptide 75, peptide 79, peptide 80, peptide 81, peptide 82, peptide 83,peptide 84, peptide 85, peptide 86, peptide 87, peptide 92, peptide 93,peptide 94, peptide 95, peptide 96, peptide 97, peptide 98, peptide 99,peptide 100, peptide 106, peptide 107, peptide 108, peptide 109, peptide110, peptide 111, peptide 112, peptide 113, peptide 114, peptide 122,peptide 123, peptide 124, peptide 125, peptide 126, peptide 127, peptide128, peptide 129, peptide 139, peptide 140, peptide 141, peptide 142,peptide 143, peptide 144, peptide 145, peptide 157, peptide 158, peptide159, peptide 160, peptide 161, peptide 162, peptide 176, peptide 177,peptide 178, peptide 179, peptide 180, peptide 196, peptide 197, peptide198, peptide 199, peptide 217, and peptide 219 as described herein; and,

(b) an amino acid sequence having between 80% to 96% sequence identityto the sequence defined in (a);

wherein the amine of the N-terminal amino acid of the mucinhypersecretion-inhibiting peptide amino acid sequence is optionallycovalently bonded by an amide bond to a carboxylic acid selected fromthe group consisting of myristic acid and acetic acid, and

wherein the carboxyl of the C-terminal amino acid of the mucinhypersecretion-inhibiting peptide amino acid sequence is optionallycovalently bonded by an amide bond to ammonia.

The peptides may be administered locally or systemically (e.g., in thefrom of a pharmaceutical composition comprising a peptide of theinvention and a pharmaceutically acceptable carrier) and may beadministered to any part of a mammal's body, including, but not limitedto, those parts of the body that produce mucus and/or mucin (e.g.,preferably to respiratory passages, nasal cavity, oral cavity, trachea,lungs, gastrointestinal tract, eye, reproductive tract, etc.). Thepeptides may be administered in various ways, including, but not limitedto, topical administration, parenteral administration, rectaladministration, pulmonary administration, nasal administration,inhalation, insufflation, and oral administration. Pulmonaryadministration may be accomplished, for example, using an aerosolizer, anebulizer, a dry powder inhaler, a metered dose inhaler, and the like.

The peptides may be prepared and administered as pharmaceuticalformulations suitable for any pharmaceutically effective administrationroute. The peptides of the invention (or pharmaceutical formulationsthereof) may be in a form suitable for oral use (for example as tablets,lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions,dispersible powders or granules, syrups or elixirs), for topical use(for example as creams, ointments, gels, or aqueous or oily solutions orsuspensions), for administration by inhalation (for example as a finelydivided powder or a liquid aerosol), for administration by insufflation(for example as a finely divided powder) or for parenteraladministration (for example as a sterile aqueous or oily solution forintravenous, subcutaneous, or intramuscular dosing or as a suppositoryfor rectal dosing).

The peptides may be administered to the airways of a mammal by anysuitable method, including generating an aerosol that includes thepeptide. Such an aerosol may be a solid or liquid, and may beaqueous-based. Suitable size particles of such an aerosol may beproduced in various ways known in the art. Particles of such an aerosolare typically less than 50 micrometers in size, preferably in the rangeof about 0.1 micrometers to about 10 micrometers in size, morepreferably in the range of about 1 micrometer to about 10 micrometers insize, and even more preferably in the range of about 2 to about 7micrometers in size, and preferably about 2 to about 7 micrometers.

Nasal administration of a peptide (or a pharmaceutical formulation of apeptide) may be accomplished, for example, by spray application of anaqueous solution or suspension of a peptide or by application of anaqueous solution or suspension of a peptide as a drop or droplet such asfrom a dropper or pipette, for example in the form of a pharmaceuticallyacceptable, preferably sterile, saline (NaCl) solution which may beoptionally buffered. Sterilization of an aqueous solution may beaccomplished, for example, by sterile filtration of an aqueous solutioncomprising the peptide, optionally in the presence of pharmaceuticallyacceptable additives such as preservatives, antioxidants, bufferingsalts, tonicity modifiers, and the like. Sterilization of an aqueoussuspension comprising the peptide may be accomplished, for example, byirradiation or sterile filtration when the suspended material is smallerin particle size than 0.2 micrometers, for example a suspensioncomprising a micelle, a liposome such as a phospholipid-stabilizedliposome, or similar particle which can pass through a 0.2 micrometerfilter. An aqueous solution (e.g., an isotonic saline solution orhypertonic saline solution, and optionally a sugar which will notchemically react with the peptide) of a therapeutically effective amountof a peptide of the invention can be sterilized, for example by sterilefiltration, placed as an aliquot containing a therapeutically effectiveamount of peptide into a sterile vial, lyophilized to form a driedpowder, and sealed, preferably in the presence of an inert atmosphere orgas, using a sterile stopper or cap. Prior to use, the dried powder canbe rehydrated by the addition of sufficient water to form an isotonicsolution as a single dose which can be administered as an aerosol to theairway of a mammal.

The amount of peptide or phainiaceutical formulation thereofadministered to a mammal may vary depending upon multiple factorsincluding, but not limited to, the particular peptide, the type ofmammal, the mammal's degree of illness, the mammal's weight, and themammal's age. In addition, it may be desirable to maintain some level ofmucin secretion/mucus production (e.g., a normal or basal level) in amammal being treated with a peptide. In one embodiment, a human is thepreferred mammal in need of treatment, and the amount of peptide that isadministered is sufficient to inhibit mucin hypersecretion which isadministered in single or multiple dose. The single dose of peptide ofthe present invention may range from 0.1 mg to about 30 mg/kg bodyweight, preferably from about 0.1 mg to about 15 mg/kg body weight, andmore preferably from about 0.1 mg to about 7.5 mg/kg body weight. Thenumber of doses and duration of treatment is dependent upon thesubject's response to treatment. Adjustments as to the amount of singledose, number of doses and duration of the treatment can be determined bythe health care provider dependent upon the symptoms and severity ofthese symptoms.

In one embodiment, an isolated peptide consists of less than 24 aminoacids and has an amino acid sequence selected from the group consistingof (a) an amino acid sequence having from 4 to 23 contiguous amino acids(or, in other embodiments, from 8 to 14 contiguous amino acids or from10 to 12 contiguous amino acids) of the reference sequence PEPTIDE 1,wherein the amino acid sequence begins from the N-terminal amino acid ofthe reference sequence; (b) a sequence substantially similar to theamino acid sequence defined in (a); (c) an amino acid sequence havingfrom 4 to 23 contiguous amino acids (or, in other embodiments, from 8 to14 contiguous amino acids or from 10 to 12 contiguous amino acids) ofthe reference sequence PEPTIDE 1, wherein the amino acid sequence endsat the C-terminal amino acid of the reference sequence; and (d) asequence substantially similar to the amino acid sequence defined in(c). The N-terminal amino group of the N-terminal amino acid and/or theC-terminal carboxyl group of the C-terminal amino acid may optionally bechemically modified as follows:

(1) the N-terminal amine group of the N-terminal amino acid of thepeptide is optionally in the form of an amide selected from the groupconsisting of:

an amide of a C₂ to C₂₄ aliphatic carboxylic acid,

an amide of trifluoroacetic acid,

an amide of benzoic acid, and

an amide of a C₁ to C₂₄ aliphatic alkyl sulfonic acid; or

the N-terminal amine group of the N-terminal amino acid of the peptideis optionally alkylated with a group selected from the group consistingof:

a C₁ to C₂₄ aliphatic alkyl group,

a linear 2-(C₁ to C₂₄ aliphatic alkyl)oxyethyl group,

an omega-methoxy-poly(ethyleneoxy)_(n)-ethyl group, where n is from 0 to10; and

(2) the C-terminal carboxylic acid group of the C-terminal amino acid ofthe peptide is optionally in the form of an amide selected from thegroup consisting of:

an amide of ammonia,

an amide of a C₁ to C₂₄ aliphatic alkyl amine,

an amide of a hydroxyl-substituted C₂ to C₂₄ aliphatic alkyl amine,

an amide of a linear 2-(C1 to C24 aliphatic alkyl)oxyethylamine group,and

an amide of an omega-methoxy-poly(ethyleneoxy)_(n)-ethylamine group,where n is from 0 to 10; or

the C-terminal carboxylic acid group of the C-terminal amino acid of thepeptide is optionally in the form of an ester selected from the groupconsisting of:

an ester of a C₁ to C₂₄ aliphatic alkyl alcohol,

an ester of a 2-(omega-methoxy-poly(ethyleneoxy)_(n))-ethanol group,where n is from 0 to 10.

The peptide has a mucin release-inhibiting effect when administered to amammal in a mucin-inhibiting amount. The peptide may also have a greatermucin-inhibiting effect on a mammal than MANS peptide when administeredat equal concentrations and/or greater aqueous solubility than MANSpeptide.

In another embodiment, an isolated peptide consists of less than 24amino acids and has an amino acid sequence consisting of a variant of anamino acid sequence having from 4 to 23 contiguous amino acids (or, inother embodiments, from 8 to 14 contiguous amino acids or from 10 to 12contiguous amino acids) of the reference sequence PEPTIDE 1, wherein theamino acid sequence begins from the N-terminal amino acid of thereference sequence or wherein the amino acid sequence ends at theC-terminal amino acid of the reference sequence. The N-terminal aminogroup of the N-terminal amino acid and/or the C-terminal carboxyl groupof the C-terminal amino acid may optionally be chemically modified asfollows:

(1) the N-terminal amine group of the N-terminal amino acid of thepeptide is optionally in the form of an amide selected from the groupconsisting of:

an amide of a C₂ to C₂₄ aliphatic carboxylic acid,

an amide of trifluoroacetic acid,

an amide of benzoic acid, and

an amide of a C₁ to C₂₄ aliphatic alkyl sulfonic acid; or

the N-terminal amine group of the N-terminal amino acid of the peptideis optionally alkylated with a group selected from the group consistingof:

a C₁ to C₂₄ aliphatic alkyl group,

a linear 2-(C₁ to C₂₄ aliphatic alkyl)oxyethyl group,

an omega-methoxy-poly(ethyleneoxy)_(n)-ethyl group, where n is from 0 to10; and

(2) the C-terminal carboxylic acid group of the C-terminal amino acid ofthe peptide is optionally in the form of an amide selected from thegroup consisting of:

an amide of ammonia,

an amide of a C₁ to C₂₄ aliphatic alkyl amine,

an amide of a hydroxyl-substituted C₂ to C₂₄ aliphatic alkyl amine,

an amide of a linear 2-(C1 to C24 aliphatic alkyl)oxyethylamine group,and

an amide of an omega-methoxy-poly(ethyleneoxy)_(n)-ethylamine group,where n is from 0 to 10; or

the C-terminal carboxylic acid group of the C-terminal amino acid of thepeptide is optionally in the form of an ester selected from the groupconsisting of:

an ester of a C₁ to C₂₄ aliphatic alkyl alcohol,

an ester of a 2-(omega-methoxy-poly(ethyleneoxy)_(n))-ethanol group,where n is from 0 to 10.

The peptide has a mucin-inhibiting effect when administered to a mammalin a mucin-inhibiting amount. The peptide also has a greatermucin-inhibiting effect on a mammal than MANS peptide when administeredat equal concentrations and/or has greater aqueous solubility than MANSpeptide.

In a further embodiment, a method of inhibiting mucin hypersecretion ina mammal comprises administering to the mammal a mucin-inhibiting amountof an isolated peptide that inhibits mucin secretion. The isolatedpeptide consists of less than 24 amino acids and has an amino acidsequence selected from the group consisting of (a) an amino acidsequence having from 4 to 23 contiguous amino acids (or, in otherembodiments, from 8 to 14 contiguous amino acids or from 10 to 12contiguous amino acids) of the reference sequence PEPTIDE 1, wherein theamino acid sequence begins from the N-terminal amino acid of thereference sequence; (b) a sequence substantially similar to the aminoacid sequence defined in (a); (c) an amino acid sequence having from 4to 23 contiguous amino acids (or, in other embodiments, from 8 to 14contiguous amino acids or from 10 to 12 contiguous amino acids) of thereference sequence PEPTIDE 1, wherein the amino acid sequence ends atthe C-terminal amino acid of the reference sequence; and (d) a sequencesubstantially similar to the amino acid sequence defined in (c). TheN-terminal amino group of the N-terminal amino acid and/or theC-terminal carboxyl group of the C-terminal amino acid may optionally bechemically modified as follows:

(1) the N-terminal amine group of the N-terminal amino acid of thepeptide is optionally in the form of an amide selected from the groupconsisting of:

an amide of a C₂ to C₂₄ aliphatic carboxylic acid, an amide oftrifluoroacetic acid,

an amide of benzoic acid, and an amide of a C₁ to C₂₄ aliphatic alkylsulfonic acid; or

the N-terminal amine group of the N-terminal amino acid of the peptideis optionally alkylated with a group selected from the group consistingof:

a C₁ to C₂₄ aliphatic alkyl group, a linear 2-(C₁ to C₂₄ aliphaticalkyl)oxyethyl group,

an omega-methoxy-poly(ethyleneoxy)_(n)-ethyl group, where n is from 0 to10; and

(2) the C-terminal carboxylic acid group of the C-terminal amino acid ofthe peptide is optionally in the form of an amide selected from thegroup consisting of:

an amide of ammonia, an amide of a C₁ to C₂₄ aliphatic alkyl amine,

an amide of a hydroxyl-substituted C₂ to C₂₄ aliphatic alkyl amine,

an amide of a linear 2-(C1 to C24 aliphatic alkyl)oxyethylamine group,and

an amide of an omega-methoxy-poly(ethyleneoxy)_(n)-ethylamine group,where n is from 0 to 10; or the C-terminal carboxylic acid group of theC-terminal amino acid of the peptide is optionally in the form of anester selected from the group consisting of:

an ester of a C₁ to C₂₄ aliphatic alkyl alcohol,

an ester of a 2-(omega-methoxy-poly(ethyleneoxy)_(n))-ethanol group,where n is from 0 to 10.

The peptide may have a greater mucin-inhibiting effect on a mammal thanMANS peptide when administered at equal concentrations and/or greateraqueous solubility than MANS peptide.

The peptides of the invention may be prepared by any suitable method,including solid-phase peptide synthesis techniques such as, for example,using fluorenylmethyloxycarbonyl (FMoc) chemistry and a suitable peptidesynthesizer such as a CS-Bio Peptide Synthesizer, or usingtert-butyoxycarbonyl (Boc) chemistry and a suitable peptide synthesizersuch as an ABI 430A Peptide Synthesizer. Protected amino acids suitablefor use in either FMoc or Boc synthesis are commercially available, forexample from Calbiochem, a unit of EMD Biosciences, San Diego, Calif. Insolid phase peptide synthesis, the C-terminal carboxyl group of thedesired peptide as an N-alpha-protected amino acid is covalently boundto a polymer support. The N-alpha-amino protecting group is then removedand a second N-alpha-protected amino acid is coupled to the attachedamino acid by formation of an amide bond to the deprotectedN-alpha-amine of the amino acid linked to the resin. These steps arerepeated with the respective protected amino acids of the desiredpeptide sequence until the desired sequence is obtained. At the end ofthe synthesis, the bond between the C-terminal amino acid and thepolymer support is cleaved to liberate the peptide. The peptide can beisolated and purified by HPLC. Useful HPLC purification methods includeion exchange chromatography and reverse phase chromatography. Solutionsof peptides can be evaporated or lyophilized to provide isolated peptidein solid form. Peptides containing oxidizable groups such as methionine,cysteine, tryptophan, residues are preferably maintained in anoxygen-free atmosphere, and, when formulated and stored in solution orsuspension, used in oxygen-free solvents.

Coupling of activated ester to the amine end of the resin-linked peptideduring synthesis can be done, for example, using an excess such as a4-fold excess of amino acid andbenzotriazol-1-yl-oxy-tris-dimethylamino-phosphoniumhexafluorophosphate, and an excess such as a 6-fold excess ofN,N-diisopropylethylamine. Peptides from Fmoc synthesis are cleaved fromthe resin with trifluoroaceticacid/thioanisole/triisopropylsilane/methanol (for example at a ratio of90:5:2.5:2.5, vol/vol/vol/vol) at 20° C. for 4 h and those from Bocsynthesis with for example HF/anisole (9:1, vol/vol) at 4° C. for 1 h.

In an Fmoc peptide strategy, the first Fmoc amino acid is attached to aninsoluble support resin via an acid labile linker. Deprotection of Fmocis accomplished by treatment of the amino acid with a base such aspiperidine. A second Fmoc amino acid is coupled utilizing apre-activated species or in situ activation (coupling reactions can bedone in situ using activating reagents known in peptide chemistry suchas DCC, HBTU, TBTU, BOP, BOP-Cl, and the like). After the desiredpeptide is synthesized, the resin bound peptide is deprotected anddetached from the solid support by acidolysis cleavage with weak acidssuch as trifluoroacetic acid (TFA) or TMSBr in the presence of ascavenger such as a thiol compound, phenol, and water, for example. Inone aspect, prior to deprotection of side chain functionality andcleavage from the resin, the terminal amine group of the peptide can betreated with a carboxylic (e.g., aliphatic carboxylic, trifluoroacetic,benzoic and the like), for example, with an aliphatic carboxylic acidspecies such as an activated form of an aliphatic carboxylic acid suchas a pentafluoroester in a manner analogous to the formation of apeptide bond to form an amide of the carboxylic or with an aliphaticsulfonic acid species (such as a sulfonyl chloride) to form asulfonamide at the N-terminal of the peptide. Alternatively, theN-terminal amine can be alkylated with, for example, an aliphaticalkylating agent (e.g., an aliphatic mesylate or tosylate) obtained byreaction of the corresponding sulfonyl chloride and a base such aspyridine with an aliphatic alcohol, which alcohol can be obtained byreduction (e.g., by lithium aluminum hydride) of an aliphatic carboxylicacid. In another aspect, corresponding D-amino acids (e.g., up to fourD-amino acids) with the remainder as L-amino acids can be used in thepeptide synthesis procedure to provide a peptide of the invention whichcan be optionally chemically modified as above. In another aspect, whenthe desired peptide amino acid sequence is formed, for example by solidphase synthesis, and the side chain protecting groups have been selectedto withstand the ester cleaving step to liberate the peptide from theresin, a side-chain protected peptide having a free carboxylic acid atthe C-terminal end of the peptide is formed. The C-terminal carboxylicacid can be converted to an active ester (e.g. a pentafluorophenylester) and treated with an amine such as ammonia to form an amide(represented as peptide-C(O)—NH₂), or with an aliphatic amine asdescribed above to form an aliphatic amide of the peptide, and anyremaining protecting groups can be removed to provide the desiredpeptide of the invention. Additionally, a C-terminal carboxylic estercan be formed from a C-terminal carboxylic acid and an aliphatic alcoholby dehydrative coupling such as by use of a carbodiimide reagent. Acidfunctional group-containing amino acids such as aspartic acid andglutamic acid can be converted to amides and esters in a fashionanalogous to the above method, and the epsilon amine group in lysine canbe converted to amides and aliphatic amines as described above forchemistry on the terminal amino group.

Examples of protected amino acids that are useful in FMoc solid phasesynthesis of peptides of this invention include the followingnon-limiting examples: N-alpha-Fmoc-L-alanine pentafluorophenyl ester;N-alpha-Fmoc-N-alpha-(2-Fmoc-oxy-4-methoxybenzyl)-alaninepentafluorophenyl ester; N-alpha-Fmoc-glycine pentafluorophenyl ester;N-alpha-Fmoc-N-alpha-(2-Fmoc-oxy-4-methoxybenzyl)-glycinepentafluorophenyl ester; N-alpha-Fmoc-L-glutamic acidg-2-phenylisopropyl ester; N-alpha-Fmoc-L-glutamic acidalpha-4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino}benzylester; N-alpha-Fmoc-L-glutamic acidgamma-4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino}benzylester; N-alpha-Fmoc-L-glutamic acid alpha-allyl ester;N-alpha-Fmoc-L-glutamic acid g-benzyl ester; N-alpha-Fmoc-L-glutamicacid alpha-t-butyl ester; N-alpha-Fmoc-L-glutamic acid gamma-t-butylester; N-alpha-Fmoc-L-glutamic acid gamma-t-butyl esterpentafluorophenyl ester; N-alpha,epsilon-Di-Fmoc-L-lysinepentafluorophenyl ester; N-alpha,epsilon-di-Fmoc-L-lysine;N-alpha-1-(4,4-Dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-N-epsilon-Fmoc-L-lysine;N-alpha-Fmoc-L-lysine;N-alpha-Fmoc-N-alpha-(2-Fmoc-oxy-4-methoxybenzyl)-N-epsilon-t-butoxycarbonyl-L-lysine;N-alpha-Fmoc-N-epsilon-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl-L-lysine;N-alpha-Fmoc-N-epsilon-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-L-lysine;N-alpha-Fmoc-N-epsilon-2-chloro-CBZ-L-lysine;N-alpha-Fmoc-N-epsilon-4-methyltrityl-L-lysine;N-alpha-Fmoc-N-epsilon-acetyl-L-lysine;N-alpha-Fmoc-N-epsilon-benzyloxycarbonyl-L-lysine;N-alpha-Fmoc-N-epsilon-t-Boc-L-lysine;N-alpha-Fmoc-N-epsilon-t-Boc-L-lysine pentafluorophenyl ester;N-alpha-Fmoc-N-epsilon-trifluoroacetyl-L-lysine;N-alpha-Fmoc-4-chloro-L-phenylalanine;N-alpha-Fmoc-4-cyano-L-phenylalanine;N-alpha-Fmoc-4-fluoro-L-phenylalanine;N-alpha-Fmoc-4-nitro-L-phenylalanine; N-alpha-Fmoc-L-phenylalanine;N-alpha-Fmoc-L-phenylalanine pentafluorophenyl ester;N-alpha-Fmoc-N-alpha-(2-Fmoc-oxy-4-methoxybenzyl)-phenylalanine;N-alpha-Fmoc-N-alpha-methyl-L-phenylalanine; N-alpha-Fmoc-L-prolinepentafluorophenyl ester;N-alpha-Fmoc-gamma-(4,4′-dimethoxybenzhydry)-L-glutamine;N-alpha-Fmoc-gamma-trityl-L-glutamine pentafluorophenyl ester;N-alpha-Fmoc-L-glutamine; N-alpha-Fmoc-L-glutamine pentafluorophenylester; N-alpha-Fmoc-N-gamma-trityl-L-glutamine;N-alpha-Fmoc-N^(G)-(4-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginineN-methoxy-N-methyl amide;N-alpha-Fmoc-N^(G)-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine;N-alpha-Fmoc-N^(G)-2,2,5,7,8-pentamethylchroman-6-sulfonyl-L-arginine;N-alpha-Fmoc-N^(G)-4-methoxy-2,3,6-trimethylbenzenesulfonyl-L-arginine;N-alpha-Fmoc-N^(G)-4-methoxy-2,3,6-trimethylbenzenesulfonyl-L-argininepentafluorophenyl ester; N-alpha-Fmoc-N^(G)-nitro-L-arginine;N-alpha-Fmoc-N^(G)-tosyl-L-arginine;N-alpha-Fmoc-O-(2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-alpha-D-galactopyranosyl)-L-serine;N-alpha-Fmoc-L-serine; N-alpha-Fmoc-O-benzyl-L-phosphoserine;N-alpha-Fmoc-O-benzyl-L-serine; N-alpha-Fmoc-O-t-butyl-L-serine;N-alpha-Fmoc-O-t-butyl-L-serine N-hydroxysuccinimide;N-alpha-Fmoc-O-trityl-L-serine; N-alpha-Fmoc-L-threonine;N-alpha-Fmoc-O-benzyl-L-phosphothreonine;N-alpha-Fmoc-O-benzyl-L-threonine; N-alpha-Fmoc-O-t-butyl-L-threonine;N-alpha-Fmoc-O-trityl-L-threonine;N-alpha-Fmoc-O-(2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-alpha-D-galactopyranosyl)-L-threonine;N-alpha-Frnoc-L-valine; N-alpha-Fmoc-L-valine pentafluorophenyl ester;N-alpha-Fmoc-N-alpha-(2-Fmoc-oxy-4-methoxybenzyl)-valine;N-alpha-Fmoc-N-alpha-(2-Fmoc-oxy-4-methoxybenzyl)-valinepentafluorophenyl ester; N-alpha-Fmoc-N-alpha-methyl-L-valine;N-alpha-Fmoc-O-(bis-dimethylamino-phosphono)-L-tyrosine;N-alpha-Fmoc-L-tyrosine; N-alpha-Fmoc-O-2,6-dichlorobenzyl-L-tyrosine;N-alpha-Fmoc-O-2-bromo-CBZ-L-tyrosine;N-alpha-Fmoc-O-2-chlorotrityl-L-tyrosine;N-alpha-Fmoc-O-benzyl-L-phosphotyrosine;N-alpha-Fmoc-O-methyl-L-tyrosine; N-alpha-Fmoc-O-phospho-L-tyrosine;N-alpha-Fmoc-O-t-butyl-L-tyrosine; N-alpha-Fmoc-O-t-butyl-L-tyrosinepentafluorophenyl ester; N-alpha-Fmoc-L-aspartic acid beta-1-adamantylester; N-alpha-Frnoc-L-aspartic acid beta-2-adamantyl ester;N-alpha-Fmoc-L-aspartic acid beta-2-phenylisopropyl ester;N-alpha-Fmoc-L-aspartic acidbeta-4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino}benzylester; N-alpha-Fmoc-L-aspartic acidalpha-4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino}benzylester; N-alpha-Fmoc-L-aspartic acid alpha-allyl ester;N-alpha-Fmoc-L-aspartic acid beta-benzyl ester; N-alpha-Fmoc-L-asparticacid alpha-t-butyl ester; N-alpha-Fmoc-L-aspartic acid beta-t-butylester; N-alpha-Fmoc-L-aspartic acid beta-t-butyl ester pentafluorophenylester; N-alpha-Fmoc-L-aspartic acid-beta-1-adamantyl esterpentafluorophenyl ester; N-alpha-Fmoc-L-aspartic acid-beta-2-adamantylester pentafluorophenyl ester; N-alpha-Fmoc-L-leucine;N-alpha-Fmoc-L-leucine pentafluorophenyl ester;N-alpha-Fmoc-N-alpha-(2-Fmoc-oxy-4-methoxybenzyl)-leucine;N-alpha-Fmoc-N-alpha-(2-Fmoc-oxy-4-methoxybenzyl)-leucinepentafluorophenyl ester; N-alpha-Fmoc-N-alpha-methyl-L-leucine;N-alpha-Fmoc-L-isoleucine; N-alpha-Fmoc-L-isoleucine pentafluorophenylester; N-alpha-Fmoc-N-alpha-methyl-L-isoleucine;N-alpha-Fmoc-beta-2,4,6-trimethoxybenzyl-L-asparagine;N-alpha-Fmoc-beta-trityl-L-asparagine pentafluorophenyl ester;N-alpha-Fmoc-L-asparagine; N-alpha-Fmoc-L-asparagine pentafluorophenylester;N-alpha-Fmoc-N-beta-(3,4,6-tri-O-acetyl-2-(acetylamino)-deoxy-2-beta-glucopyranosyl)-L-asparagine;N-alpha-Fmoc-N-beta-trityl-L-asparagine;N-alpha-Fmoc-N-im-methyltrityl-L-histidine;N-alpha-Fmoc-N-im-t-Boc-L-histidine cyclohexylammonium salt;N-alpha-Fmoc-N-im-tosyl-L-histidine;N-alpha-Fmoc-N-im-trityl-L-histidine;N-alpha-Fmoc-S-acetamidomethyl-L-cysteine;N-alpha-Fmoc-S-acetamidomethyl-L-cysteine pentafluorophenyl ester;N-alpha-Fmoc-S-benzyl-L-cysteine;N-alpha-Fmoc-S-p-methoxybenzyl-L-cysteine;N-alpha-Fmoc-S-p-methoxytrityl-L-cysteine;N-alpha-Fmoc-S-t-butyl-L-cysteine; N-alpha-Fmoc-S-t-butyl-L-cysteinepentafluorophenyl ester; N-alpha-Fmoc-S-t-butylthio-L-cysteine;N-alpha-Fmoc-S-t-butylthio-L-cysteine pentafluorophenyl ester;N-alpha-Fmoc-S-trityl-L-cysteine; N-alpha-Fmoc-S-trityl-L-cysteinepentafluorophenyl ester. These and other amino acid reagents for use insolid phase synthesis of peptides are commercially available, forexample, from Calbiochem Corporation. Aliphatic carboxylic acids arealso available from Sigma-Aldrich Chemical Company.

The peptides may also be produced by a solid phase synthesis using a Bocstrategy, wherein a first Boc amino acid is attached to an insolublesupport resin via a HF cleavable linker. Deprotection by removal of theBoc group is accomplished by treatment of the Boc-amino acid with TFA. Asecond Boc amino acid is then coupled utilizing a pre-activated speciesor in situ activation. After the desired peptide is synthesized, theresin-bound peptide is deprotected and detached from the solid supportvia cleavage using a strong acid such as HF, TFMSOTf, or TMSOTf. Anadditive such as a thiol compound is added to protect the peptide fromany carbocations generated during cleavage. The following protectinggroups are compatible with HF cleavage: Arg(Mts); Cys(4-MeOBzl); His(Z);Arg(Tos); Glu(OBzl); Lys(Cl—Z); Asp(OBzl); Glu(OcHex); Ser(Bzl);Asp(OcHex); His(Bom); Thr(Bzl); Cys(Acm); His(Dnp); Trp(CHO);Cys(4-MeBzl); His(Tos); Tyr(Br-Z); Asp(OtBu); His(Trt). The followingprotecting groups are compatible with TFMSOTf cleavage: Arg(Mts);His(Bom); Met(O); Asp(OBzl); His(Dnp)); Ser(Bzl); Cys(Acm); His(Tos);Thr(Bzl); Cys(4-MeBzl); His(Z); Trp(CHO); Glu(OBzl); Lys(Cl—Z);Tyr(Br—Z). The following protecting groups are compatible with TMSOTfcleavage: Arg(Mts); Glu(OcHex); Trp(CHO); Arg(Mbs); His(Bom); Trp(Mts);Asp(OBzl); Lys(Cl—Z); Tyr(Br—Z); Asp(OcHex); Met(O); Tyr(Bzl); Cys(Acm);Ser(Bzl); Tyr(Cl-Bzl); His(Bom); Thr(Bzl). Coupling of amino acidcarboxylic acids and amines to form peptide amide bonds can beaccomplished in a Boc strategy using carbodiimides such asdicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), t-butylmethyl- and t-butylethyl-carbodiimides; BOP; PyBroP; PyBOP; HBTU; TBTU;and HATU, all of which reagents require bases for activation and whichact by the formation of symmetrical anhydride. Alternatively,carboxylic-carbonate or carboxylic-phosphinic mixed anhydride reagents,prepared by reacting isobutyl- or isopropyl-chloroformate andsubstituted phosphinic chlorides with the N-alpha-protected amino acid,or N-carboxyanhydrides (NCA's) or Leuchs' anhydride can be used. As withFmoc chemistry, the N-terminal amine and the C-terminal carboxylic acidgroup can be chemically modified according to synthetic strategy outlineabove.

EXAMPLES

The invention will be further explained by the following illustrativeexamples that are intended to be non-limiting. These examples providethe results from testing of specific peptides that are representative ofpeptides disclosed in the application. The peptides are listed with anymodifications to the N-terminal amino group of the N-terminal amino acidand/or the C-terminal carboxyl group of the C-terminal amino acid on theleft- and right-hand sides, respectively, of the amino acid sequence,which is indicated by a Peptide # of the test peptides. Table IXcontains the key to the peptide's sequence. Myr- and Ac- areabbreviations of myristoyl and acetyl, respectively, which arecovalently bonded in amide bonds to the N-terminal amino acid in therespective peptide amino acid sequences; an —NH2 denotes an amide ofammonia which is covalently bound to the C-terminal end of the peptideamino acid sequence.

Example 1A Relative Efficacy of Test Peptides in a Mouse Model of AsthmaI. Protocols and Methods

Experiments were designed to test whether or not the MANS peptide andother test peptides related thereto inhibit hypersecretion of mucin inmurine airway in vivo. The ovalbumin-sensitized mouse model of allergicinflammation and asthma used in these studies was as described by Singeret al. (2004), supra. As a negative control, a control peptidecontaining an N-terminal myristoyl group and the same amino acids asMANS peptide but arranged in random order (i.e., the random N-terminalsequence, RNS, myristoyl-peptide 232) was tested alongside the proposedactive peptides. BP2 mice, aged 6-8 wks, were immunized subcutaneouslytwice at weekly intervals with 1 μg of ovalbumin. After 14 days ofsensitization, the animals were exposed to aerosolized ovalbumin, whichcauses a pronounced goblet cell hyperplasia after 72 hours. At the 72 hrtime point, the secretagogue, methacholine (60 mM) was delivered using aBuxco system nebulizer providing a fine aerosol for 90 seconds. Fifteenmin prior to the secretagogue challenge, 50 μL of the test peptide, at 3different concentrations (10 μM, 100 μM, or 140 μM), was administered byintratracheal route. The RNS peptide was tested at the highest doselevel only (50 μL, of 140 μM solution). The MANS as well as RNS peptidewere freely soluble in 120 mM sodium acetate, pH 7. The various controlsused in these experiments are tabulated in Table I below. Eachexperiment was carried out in 6 mice per point, and each set ofexperiments was repeated 3 times. To test strain-to-strain variations,the above experiment was repeated in Balb/C mice under similar protocol.Both stimulated and unstimulated mucin secretion, in mice treated with120 mM sodium acetate (data not shown), were identical to salinecontrol. Following the methacholine challenge, the animals weresacrificed and bronchoalveolar lavage (BAL) performed on 6 animals pergroup for analysis of secreted mucin.

Table I indicates the general protocol for the experiment.

TABLE I General protocol for methacholine-induced mucin hypersecretionin the presence of test peptide # Secretagogue Test Group mice TreatmentPeptides Challenge Saline control 6 Endotoxin-free 0.9% NaCl None YesSaline control 6 Endotoxin-free 0.9% NaCl None No Test peptide 6Endotoxin-free 0.9% NaCl + Each test Yes 140 μM Each test peptidepeptide Test peptide 6 Endotoxin-free 0.9% NaCl + Each test Yes 100 μMEach test peptide peptide Test peptide 6 Endotoxin-free 0.9% NaCl + Eachtest No 100 μM Each test peptide peptide Test peptide 6 Endotoxin-free0.9% NaCl + Each test Yes 10 μM Each test peptide peptide Test peptide 6Endotoxin-free 0.9% NaCl + Each test No 10 μM Each test peptide peptideNegative (control) 6 Endotoxin-free 0.9% NaCl + RNS Yes peptide (RNS)140 μM RNS peptide

II. Analyses of BAL Fluid

Mice were sacrificed by cervical dislocation, followed by rapid exposureof the trachea and insertion of a cannula through a small incision.Bronchoalveolar lavage (BAL) fluid was collected with 0.5 ml followed by3×1 ml PBS containing PMSF (5 mM), EDTA (5 mM), and DTT (5 mM). BALfluid was separated into cell-free and cell fractions via briefcentrifugation prior to any analyses being performed. The cell-freesupernatants were analyzed for the presence of mucin via an ELISA methodusing anti-mucin antibodies shown to react with mouse mucin.Specifically, a mouse anti-MUC5AC antibody that recognizes thecarbohydrate portion of secreted mucin was used in these assays. Datagenerated from each BAL sample were normalized to total protein asdetermined by Bradford assay. Mucin content was expressed as the valueobtained with the anti-mucin antibody minus that obtained with thenon-immune control antibody. All ELISA data were statistically examinedusing a one-way ANOVA. The experimental data were consideredsignificantly different from control when the p<0.05.

Tables II, III, IV, V and VI below summarize the effect of variouspeptides on mucin secretion in Balb/C mice and BP2 mice. As shown inTable II, at 100 μM of the various test peptides, mucin secretion wasbetween 8% and 56% of that of the control (i.e., no peptide).

TABLE II Mucin Secretion in a Murine Model of Asthma (Experiments inBalb/C Mice) Peptide % Mean (peptide #/identifier) control error Nopeptide 100 4.2 MANS, myr-peptide 1 33.8 5.8 RNS, myr-peptide 232 96 5.2myr-peptide 79 50 10.8 myr-peptide 233 21 14.2 myr-peptide 234 8 4.2myr-peptide 235 13 3.3 peptide 79 10 3.3 myr-peptide 234-NH₂ 27.8 1.6myr-peptide 106 33.6 5.8 peptide 106) 33.8 3.3 myr-peptide 106-NH₂ 29.34.2 myr-peptide 236-NH₂ 51 2.0 Ac-peptide 106 21.5 18.3 myr-peptide 13755 4.2 myr-peptide 137-NH₂ 56 3.3 All peptides tested at 100 μM. Allvalues significant vs. control (p < 0.001) except myr-peptide 137 (p <0.01) and RNS.

TABLE III Mucin Secretion in a Murine Model of Asthma (Experiments inBalb/C Mice) Peptide % Mean (peptide #/identifier) control error Nopeptide 100  8.1 MANS, myr-peptide 1 40 11.8 RNS, myr-peptide 232  81**2.8 myr-peptide 11 54 5.1 myr-peptide 37  35** 11.8 myr-peptide 79 4810.3 myr-peptide 15  38* 10.6 myr-peptide 45  35** 10.3 myr-peptide 9168 14.7 myr-peptide 153 50 7.3 All peptides tested at 100 μM. *= valuessignificant vs. control (p < 0.05); **= values significant vs. control(p < 0.01).

TABLE IV Mucin Secretion in a Murine Model of Asthma (Experiments inBalb/C Mice) Peptide % of Mean (peptide #/identifier) control error Nopeptide 100   5.3 MANS, myr-peptide 1 (100 μM) 29** 4.2 RNS, myr-peptide232 (100 μM) 108   8.5 peptide 237 (10 μM) 67*+ 8.5 peptide 237 (100 μM) 8** 3.8 myr-peptide 106 (10 μM) 74*+ 10.6 myr-peptide 106 (100 μM) 24**9.6 peptide 106 (10 μM) 64*  4.2 peptide 106 (100 μM) 40** 9.6myr-peptide 106-NH₂ (10 μM) 67*+ 4.2 myr-peptide 106-NH₂ (100 μM) 17**5.3 *= values significant vs. control (p < 0.05); **= values significantvs. control (p < 0.01). += value significant vs. 100 μM treatment (p <0.05).

TABLE V Mucin Secretion in a Murine Model of Asthma (Experiment in BP2Mice) (% of Control) Mucin Secretion (% of Control) Peptide (Conc.) 10μM 100 μM 140 μM MANS peptide 35 12 8 RNS peptide — 100 100

Experiments were designed to determine the duration of action of testpeptides in a murine model of asthma. As described in Method 1A, Balb/Cmice were immunized with ovalbumin. After 14 days, the animals werechallenged with aerosolized ovalbumin. After 72 hours the secretagogue,methacholine was delivered by aerosolization. The test peptides (50 L of100 M solution) were administered by intratracheal route 30 min, 60 min,or 120 min prior to methacholine challenge. The animals were sacrificedand BAL performed for analysis of secreted mucin The results of thisexperiment are given in Table VI.

TABLE VI Duration of Action of Test Peptides in a Murine (Experiments inBalb/C Mice) (% of Control) Peptide 30 min 60 min 120 min Control 100100 100 MANS, myr-peptide 1 7 44 50 RNS, myr-peptide 232 100 100 100Ac-peptide 106 21 72 70 peptide 106 23 48 57

An alternative mouse test model and method of quantitatively determiningmucin in mouse lungs is useful to evaluate the activity of peptides ofthe present invention. This method is described by Evans et al (Am. J.Respir. Cell Mol. Biol. Vol. 31, pp 382-394, 2004). Briefly, Balb/cfemale mice (5-8 weeks old, 20-25 g each) are sensitized weekly for fourweeks by intraperitoneal (i.p.) injection of 100 μL solution containing2.2 mg of alum salt and 20 μg of ovalbumin in normal saline. Seven daysafter the last i.p. injection, the mice are challenged by aerosoladministration over 30 minutes of a 2.5% solution of ovalbumin dissolvedin normal saline. The aerosol is generated with AeroMist CA-209compressed air nebulizer (CIS-US, Inc., Bedford, Mass.).

Three days post ovalbumin challenge, 50 μL of the test peptide isdelivered in each nostril of the mouse in 10 pit aliquots over 2-3minutes. Fifteen minutes later the mice are treated with aerosolized 100mM ATP solution over 5 minutes. After 20 minutes, the mice areanesthetized by i.p. injection of a mixture of ketamine, xylazine, andacepromazine and the lungs are perfused with saline via the rightcardiac ventricle to clear blood from the pulmonary tissues. Under deepanesthesia, animals are tracheostomized using a 20 gauge blunt tipcannula and sacrificed by exsanguination via the abdominal aorta.Fixative (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.0) isinfused intratracheally at 10-15 cm pressure and the lungs are fixed insitu for 30 minutes, removed from the thoracic cavity and fixedovernight at 4° C. Lungs are embedded in paraffin and cut into 6 μmsections.

For fluorescent labeling of mucin, tissues are stained using a periodicacid fluorescent Schiff (PAFS) staining procedure. Tissues are firstoxidized in 1% periodic acid (10 min), rinsed, treated with acriflavinefluorescent Schiff's reagent (0.5% acriflavine HCl wt/vol, 1% sodiummetabisulfite wt/vol, 0.01 N HCl) for 20 min, rinsed in double deionizedH2O, and rinsed 2×5 min in acid alcohol (0.1 N HCl in 70% ethanol).Slides are dehydrated in graded ethanol solutions and allowed to air dryin the dark. Once dry, PAFS-stained slides are coverslipped with Canadabalsam mounting medium (50% Canada balsam resin, 50% methyl salicylate;Fisher Chemicals).

For the quantitation of mucin, PAFS-stained slides are examined underthe 40× objective. Images of 10 fields from the axial bronchi arecaptured, and camera settings are managed using MagnaFire 2.1(Optronics). PAFS imaging is performed by exciting specimens using adual excitation filter (500 nm and 573 nm peaks) and observing specimensusing a dual emission filter with peaks at 531 nm (green) and 628 nm(red). For each field, an image is first generated using only the redacquisition channel on the camera (590 ms exposure). The same image wasthen recaptured using both the red and green channels on the camera (590ins red, 450 ms green). For morphometric analysis, the volume densityand fluorescence intensity of mucin staining are then measured usingImagePro Plus. Volume density of mucin staining in the airway epitheliumis calculated stereologically. Briefly, the ratio of surface area ofstaining to total surface area of the epithelium is divided by aboundary length measurement, which is a product of the total epithelialsurface area, the basement membrane length, and the geometric constant4/π. As a result, data is presented as the volume of intracellular mucincontents per surface area of the basement membrane. The mucin secretionis expressed as a fraction of total epithelial content.

Example 1B Effect of Aerosolized Ac-peptide 106 Peptide Administrationon Mucin Hypersecretion in a Mouse Model with Goblet Cell Metaplasia andAirway Obstruction

The efficacy of aerosolized Ac-peptide 106 on mucin hypersecretion in amouse model of asthma was determined at two concentrations. Briefly, 5-to 8 week old BALB/c mice were immunized weekly as described above inExample 1A for 3 weeks with ovalbumin. On Day 28 the mice werechallenged with aerosolized 2.5% ovalbumin in normal saline for 30minutes. Three days after ovalbumin challenge, each group of mice (n=2)was administered an aerosolized isotonic solution of Ac-peptide 106 at10 mM or 30 mM for 1 hour with an AeroMist CA-209 nebulizer. The massmedian aerodynamic diameter of the aerosol particles was 1.49 um (range0.4 to 4.7 um). Given these concentrations of Ac-peptide 106 and thisparticle size, the calculated pulmonary deposition of Ac-peptide 106 was0.38 mg/kg body weight and 1.1 mg/kg body weight, respectively.

Ac-peptide 106 administration was immediately followed by aerosoladministration of the secretagogue adenosine triphosphate (ATP) (100 mMin saline) for 5 minutes. The mice were sacrificed, and the lungs wereharvested within 20 minutes. The lungs were rinsed with saline, fixedwith paraformaldehyde, embedded in paraffin, and sectioned. The sectionswere quantitatively analyzed for the presence of mucin by quantitativeimmunohistochemistry according to the method of Evans et al. (Am. J.Respir. Cell Mol. Biol. Vol. 31, pp 382-394, 2004). The results indicatethat Ac-peptide 106 was effective as an inhibitor of ATP-induced mucinhypersecretion by 30 to 67% at 0.38 and 1.1 mg/kg body weight,respectively.

Example 2 Evaluation of Qualitative Solubility of Test Peptides in 0.5Normal Saline

Various test peptides (1 to 5 mg each) were weighed out accurately inindividual 2 mL screw cap glass vials and aliquots of 25 μL of 0.5 Nsaline, pH 6.5, were added at 25° C. and ambient pressure. Thesolubility was evaluated visually. If the test peptide dissolvescompletely with the first aliquot of saline, its solubility wascalculated and designated as more than the calculated amount. Thus, if3.5 mg of a test peptide dissolves instantly with the first 25 μLaliquot of 0.5 N saline, its solubility is stated as >140 mg/mL.Similarly, if 1.7 mg of a test peptide fails to dissolve in 1.7 mL of0.5 N saline, its solubility is stated as <1.0 mg/mL. The results of theevaluation of solubility of various test peptides are listed in TableVII.

The solubility of various peptides in half-normal saline can also bedetermined by one of the following two semi-quantitative methods, Method1 and Method 2. Method 1 can be used for those peptides whose solubilityin half-normal saline will be less than about 10 mg/mL, while Method 2can be used for peptides that will be soluble in half-normal saline atmore than about 30 mg/mL.

Method 1

Each peptide is dissolved in dimethylsulfoxide (DMSO) at a concentrationof 1 mg/mL. An aliquot of this solution is diluted five fold withhalf-normal saline to provide 0.2 mg/mL solution. The latter peptidesolution is subjected to HPLC analysis using C18/5 micron column. Theelution buffers consist of 0.1% trifluoroacetic acid (TFA) in water(buffer A) and 0.1% TFA in 100% acetonitrile (buffer B). The peptide iseluted with a gradient from 5% B to 45% B during about 20 min. The areaunder the curve (AUC) for each peptide peak can be thus determined. ThisAUC value can be used as a standard (AUC-Std) to determine theconcentration of each corresponding peptide in the supernatant of asaturated solution.

Saturated solution of each peptide can be prepared by adding sufficientquantity of a peptide to 1 mL of half-normal saline until a cloudysuspension is obtained. The latter is then centrifuged at 10,000×g forabout 10 min. The supernatant is then subjected to HPLC analysis. Thesupernatant is further diluted with half-normal saline, if necessary,prior to HPLC analysis. The AUC (AUC-Sat) obtained from this analysis isused to determine the concentration of the peptide in the saturatedsolution by the following formula:

Concentration of the peptide in saturated solution=AUC-Std×0.2/AUC-Sat.

Method 2

This method can provide an estimate of peptide solubility in half-normalsaline. The method consists of adding a weighed quantity (denoted as“xx” milligrams) of a peptide to dissolve in 1 mL of half-normal saline.The results are presented as >xx mg/mL, where xx is the amount ofpeptide added to the half-normal saline.

TABLE VII Peptide Solubility at 20° C. in 0.5N saline, pH 6.5 Solubilityin Sequence No. 0.5N Saline (mg/mL) myr-peptide 1 <5.0 Ac-peptide 1 >125myr-peptide 232 >15 myr-peptide 11 >2.0 myr-peptide 37 >2.0 myr-peptide79 >2.0 myr-peptide 238 >2.0 myr-peptide 233 >3.0 myr-peptide 234 >3.0myr-peptide 235 >60 peptide 79 >60 myr-peptide 79-NH₂ <1.0 myr-peptide237 <1.0 myr-peptide 237-NH₂ <1.0 peptide 237 >80 myr-peptide 234-NH₂<2.0 Ac-peptide 79-NH₂ >60 Ac-peptide 79 >100 Ac-peptide 239 >50Ac-peptide 240 N/A Ac-peptide 241 >50 myr-peptide 106 >10 peptide106 >70 myr-peptide 106-NH₂ <10 myr-peptide 236 <10 myr-peptide 236-NH₂<10 peptide 106-NH₂ >100 Ac-peptide 106 >100 cyclic-peptide 106 >150Ac-peptide 242 >100 Ac-peptide 243 >50 Ac-peptide 236 >80 Ac-peptide244 >120 Ac-peptide 245 >100 Ac-peptide 247 >100 Ac-peptide 248 >100Ac-peptide 249 <1 myr-peptide 121 <1.0 Ac-peptide 121 >20 myr-peptide137 <1.0 myr-peptide 137-NH₂ N/A Ac-peptide 250 N/A Ac-peptide 137 >200myr-peptide 15 >80 myr-peptide 45 >80 myr-peptide 91 <20 myr-peptide 153<10 myr-peptide 143 <1.0 Ac-peptide 143 >230 myr-peptide 179 <1.0Ac-peptide 179 >150 myr-peptide 219 <1.0 Ac-peptide 219 >200 Ac-peptide219-NH₂ >200 Ac-peptide 251 >200 Ac-peptide 93-NH₂ >90 Ac-peptide108-NH₂ >150 Ac-peptide 124-NH₂ >100 Ac-peptide 141-NH₂ >200 Ac-peptide159-NH₂ >200 Ac-peptide 246 <30 Ac-peptide 252 <30

Example 3 Stability of Test Peptides in Biological Fluids

Various test peptides in human plasma, human BALF, and CF patient mucuswere analyzed to determine the susceptability of the test peptide toproteolysis in the biological fluids. In addition, first order kineticanalyses were performed on samples that exhibited concentration decaysin order to determine the test peptide half-life. The samples wereanalyzed either on the same day as received or stored at −20° C. andanalyzed within the following two days.

I. Collection and Processing of Biological Fluids

A. Human Plasma:

Fresh human blood samples were collected in citrate bufferedvacuutrainers (in absence of EDTA or heparin). Red blood cells (RBC)were removed by centrifugation of the blood at 4000×g for 10 min at 4°C. Plasma aliquots (0.9 mL) were then spiked with 0.1 mL of 0.5 mg/mLsolution of the test peptide in 75 mM sodium acetate buffer at pH 7.0and incubated in a water bath maintained at 37° C. Duplicate aliquots of10 μL were withdrawn at 5, 15, 30, 60, and 180 minutes intervals andimmediately “quenched” with 990 μL of a solution consisting of 50%acetonitrile+50% water containing 0.2% formic acid. The samples werethen subjected to liquid chromatography-mass spectrometry (LCMS)analysis.

B. Human BAL Fluid:

Samples of BALF collected from COPD patients were obtained and frozen.The BALF samples were thawed, mixed together and centrifuged at 10,000×gfor 10 min at 4-8° C. The supernatant (0.9 mL) was spiked with 0.1 mL of0.5 mg/mL solution of the peptide in 75 mM sodium acetate buffer at pH7.0 and processed and analyzed as described above.

C. Human CF Mucus:

The frozen mucus (sputum) from a CF patient was thawed and mixed with 2volumes of 75 mM sodium acetate buffer, pH 7 with the help of a glasstissue grinder, centrifuged at 10,000×g for 10 min at 4-8° C. The pelletwas re-suspended in 1 volume of the acetate buffer and centrifuged. Thetwo supernatants were combined and used as follows. The supernatant (450μL) was spiked with 50 μL of 0.5 mg/mL solution of the peptide in 75 mMsodium acetate buffer at pH 7.0 and processed and analyzed as describedabove.

II. Concentration Analysis

All samples were analyzed using LC/MS/MS (MDS/SCIEX, API 4000 Model).Chromatography was performed using a Phenomenex Luna C18 column, whilemass spectrometry was performed using positive ion electrosprayionization.

A. Analysis of Peptides Ac-PEPTIDE 79-NH₂, PEPTIDE 106-NH₂, Ac-PEPTIDE106 in Human Plasma and BALF and Analysis of Peptides myr-PEPTIDE 106,PEPTIDE 106, PEPTIDE 106-NH₂, Ac-PEPTIDE 106, and cyc-PEPTIDE 106 (acyclic peptide) in Human Plasma and Human CF Mucus

Calibration standards were analyzed by LCMS at concentrations of 0.100μg/mL, 1.00 μg/mL, 10.0 μg/mL, and 100 μg/mL for each peptide, with theexception of the calibration standards for peptides myr-PEPTIDE 106,PEPTIDE 106, PEPTIDE 106-NH₂, PEPTIDE 106, and cyclic peptide,cyc-PEPTIDE 106, in human mucus, which were analyzed at concentrationsof 0.100 μg/mL, 1.00 μg/mL, 10.0 μg/mL, and 75 μg/mL. Standards wereprepared in 1% human BALF, plasma, or mucus in 50/50 water/acetonitrilemixture containing 0.2% formic acid. The instrument response values foreach set of standards were fit using a 1/(concentration)² linearleast-squares line. Sample concentrations were calculated using theslope and intercept of this line. Concentrations outside of thecalibration range were determined by extrapolating the calibrationcurve.

B. Analysis of Peptides myr-PEPTIDE 234-NH₂, myr-PEPTIDE 234, PEPTIDE106, and myr-PEPTIDE 106 in Human Plasma and BALF

Duplicate calibration standards were analyzed at a concentration of 50μg/mL for each peptide. The instrument response values for each set ofstandards were fit through an intercept of 0 using an unweighted, linearleast-squares line. Sample concentrations were calculated using theslope and intercept of this line.

C. Analysis of Peptides PEPTIDE 237, myr-PEPTIDE 106-NH₂, myr-PEPTIDE236, and myr-PEPTIDE 236-NH₂ in Human Plasma and BALF

For the analysis of peptide concentration in plasma, duplicatecalibration standards were analyzed at a concentration of 25 μg/mL and50 μg/mL for each peptide. The instrument response values for each setof standards were fit through an intercept of 0 using an unweighted,linear least-squares line. Sample concentrations were calculated usingthe slope and intercept of this line. Concentrations outside of thecalibration range were determined by extrapolating the calibrationcurve.

For the analysis of peptide concentration in BALF samples, one 50 μg/mLcalibration standard prepared in a mixture of BALF, water, acetonitrile,and formic acid was analyzed with each sample set.

III. Kinetic Profiles

First order kinetic profiles were determined using the software programWatson for all samples that exhibited a noticeable decay inconcentration. Watson fits the log transformed data with a least-squaresline to determine parameters such as C_(max) (maximum concentration),intercept, rate constant, slope, and T_(1/2) (half-life). The resultingkinetic parameters were based on the linear fit, not the actualconcentration values.

Time-zero calibration standards in solution were not included as part ofthe kinetic profiles. However, true first order kinetics do not requireinclusion of time-zero data points to accurately describe changes inconcentration as a function of time.

The half-life of the test peptides in human plasma (Plasma t_(1/2)),human BALF (BALF t_(1/2)), and human CF mucus (Mucus t₁₁₂) are listed inthe Table VIII.

TABLE VIII Plasma Plasma BALF BALF Mucus Mucus t_(1/2) t_(1/2) t_(1/2)t_(1/2) t_(1/2) t_(1/2) Peptide (hours) (hours) (hours) (hours) (hours)(hours) identifier^(#) (1^(st) exp.) (2^(nd) exp.) (1^(st) exp.) (2^(nd)exp.) (1^(st) exp.) (2^(nd) exp.) myr-peptide 234-NH₂ 3.82 — 1.70 — — —myr-peptide 234 * — 1.08 — — — peptide 106 0.28 0.15 1.88 — 0.23 0.37myr-peptide 106 1.23 0.90 1.03 — 9.53 3.00 peptide 237 0.49 — * * — —myr-peptide 106-NH₂ * — 10.52  12.81  — — myr-peptide 236 0.72 — 3.552.94 — — myr-peptide 236-NH₂ * — 4.16 4.08 — — Ac-peptide 79-NH₂ 2.64— * — — — peptide 106-NH₂ 0.30 0.13 4.00 — 0.08 0.13 Ac-peptide 106 *0.03 1.89 — 0.55 0.47 cyclic-peptide 106 — 1.04 — — 1.10 2.00 ^(#)myrand Ac are respectively myristoyl and acetyl groups covalently bonded tothe peptide at the N-terminal amine site; —NH₂ is a covalently bondedamide at the C-terminal carboxylic group of the peptide; seq no. and cycare abbreviations for sequence number and cyclic, respectively. —:Experiment not performed. *: Not enough data from experiment tocalculate half-life.

The peptide, myr-peptide 236, is myristoyl-PEPTIDE 236. All otherpeptides used in the experiment are described above.

Example 4 Efficacy of MANS Peptide on Mucus Secretion in UpperRespiratory Tract of Primates

The purpose of the experiment was to determine the ability of MANSpeptide to inhibit mucus secretion in the upper respiratory tract ofhealthy adult rhesus monkeys. The test used is a standard method forevaluation of nasal secretory activity, and non-human primates typicallyprovide a good correlation to activity in man.

Methods

A total of 17 healthy young adult male rhesus monkeys without any priorhistory of rhinitis were used for the experiment. None of the monkeyshad rhinitis before or after the study. The nasal mucus secretion ofeach monkey was measured in its left nostril prior to any treatment.This value is considered 100% mucus secretion. The monkeys were thenrandomly divided into the following 4 groups:

-   -   Group 1: Normal saline, control (n=3);    -   Group 2: Sodium acetate, solvent control (n=4);    -   Group 3: RNS peptide, negative control (n=5); and    -   Group 4: MANS peptide, test peptide (n=5).

Saline was placed in the left nostril of all 17 animals prior to anytreatment. The right nostrils were treated with 2.0 mL of either saline,sodium acetate, RNS peptide or the MANS peptide. Thus, each animal hadits own internal control. Nasal lavage was performed on both nostrils ofeach animal 1 hour after the treatment with the test articles. Alllavages were immediately frozen at −80° C. and analyzed for mucuscontent by ELISA.

A. Test Articles:

Normal saline, filter sterilized; Sodium acetate, 150 μM, filtersterilized 3. RNS Peptide—140 μM solution in 150 μM filter sterilizedsodium acetate and MANS Peptide—140 μM solution in 150 μM filtersterilized sodium acetate

B. Test Animals:

Rhesus monkeys; Number of animals: 17; Sex: all healthy males; Age: 3 to4 years; Body weight: 4 to 7 Kg, Average 5.03 Kg; AcclimatizationPeriod: 7 days; Identification Method: Unique tattoo with a 5 digit IDnumber. History: All animals were used for a vaccine study forimmunization against meningitis 12-24 months prior to this test. Allanimals also received the routine immunization against measles andtetanus during their infancy.

C. Animal Management:

Husbandry: Conditions conformed to Standard Operating Procedures, whichare based on the “Guide for the Care and Use of Laboratory Animals”.Food: Standard rhesus monkey diet was provided daily. Water: Freelyavailable, municipal water was delivered through an automatic wateringsystem. Housing: Animals were housed individually in approved stainlesssteel cages identified by a card indicating the animal numbers, testcode, sex, animal code. Environmental: The room temperature wasmonitored daily. The temperature range for the room was within a rangeof 20-26° C. The humidity range for the room, monitored daily, was40-70%. The light cycle was controlled using an automatic timer (12hours light, 12 hours dark.) Personnel: Associates involved wereappropriately qualified and trained for primates.

Results

The results provide data that the RNS peptide, sodium acetate buffer, ornormal saline did not have any effect on mucus secretion whereas mucussecretion was inhibited by almost 75% with MANS peptide.

Example 5 Tissue Culture Method for Determination of Secreted Mucin inHuman Bronchial Epithelial Cells

HBE1 is a papilloma virus-transformed human bronchial epithelial cellline capable of mucin secretion when cultured in air/liquid interface.HBE1 cells were cultured in the air/liquid interface as describedpreviously (Li et al, J. Biol. Chem., volume 276, pp 40982-40990, 2001).Briefly, HBE1 cells were cultured in air/liquid interface by seeding anappropriate number of cells in 12-well Transwell clear culture inserts(Costar, Cambridge, Mass.) that were thinly coated with rat tailcollagen, type I (Collaborative Biomedical, Bedford, Mass.). Cells wereinitially cultured submerged in medium in a humidified 95% air, 5% CO2environment for 5-7 days until nearly confluent. At that time, theair/liquid interface was created by removing the apical medium andfeeding cells basalaterally. Medium was renewed daily thereafter. Cellswere cultured for an additional 14 days to allow full differentiation.The accumulated mucin at the apical surface of the cells was removed bywashing with phosphate-buffered saline, pH 7.2. To collect the baselinemucin secretion, cells were incubated for 30 min with medium alone andsecreted mucin in the apical medium was collected, and quantitated byELISA. To determine the mucin hypersecretion induced by a mucinsecretagogue, cells were exposed to medium containing 0.5 μM phorbolmyristate acetate, (PMA) for 30 min. and mucin was collected andquantitated by ELISA. In order to determine the inhibition ofPMA-induced mucin hypersecretion by a test peptide, cells werepre-incubated with medium containing 25 or 50 μM test peptide for 15 minfollowed by 30 min incubation with 0.5 μM PMA. Six wells were used foreach test peptides and for each control. Secreted mucin in the apicalmedium was collected and quantitated by sandwich ELISA method usingalkaline phosphatase-conjugated mucin (MUC5A) specific antibody (ZymedLaboratories, San Francisco, Calif.).

Treatment of HBE1 cells with 0.5 μM PMA resulted in a 20% increase inmucin secretion. This PMA-induced increase in mucin secretion was 100%blocked by pretreatment with 25 μM MANS peptide or with 25 μM Ac-peptideno: 106. Ac-peptide no: 219 at 25 μM not only inhibited 100% ofPMA-induced mucin secretion, but also inhibited mucin secretion to alevel 20% below the unstimulated medium control. Ac-peptide no: 251 hada minimal 6% inhibitory effect on PMA-induced increase in mucinsecretion.

Table IX contains a listing of peptides of this invention and theirrespective amino acid sequences and corresponding SEQ ID NOS.

TABLE IX  Peptides and Amino Acid Sequences Peptide No. SequenceSeqence ID No. peptide 1 GAQFSKTAAKGEAAAERPGEAAVA SEQ ID NO. 1 peptide 2GAQFSKTAAKGEAAAERPGEAAV SEQ ID NO. 2 peptide 3 AQFSKTAAKGEAAAERPGEAAVASEQ ID NO. 3 peptide 4 GAQFSKTAAKGEAAAERPGEAA SEQ ID NO. 4 peptide 5AQFSKTAAKGEAAAERPGEAAV SEQ ID NO. 5 peptide 6 QFSKTAAKGEAAAERPGEAAVASEQ ID NO. 6 peptide 7 GAQFSKTAAKGEAAAERPGEA SEQ ID NO. 7 peptide 8AQFSKTAAKGEAAAERPGEAA SEQ ID NO. 8 peptide 9 QFSKTAAKGEAAAERPGEAAVSEQ ID NO. 9 peptide 10 FSKTAAKGEAAAERPGEAAVA SEQ ID NO. 10 peptide 11GAQFSKTAAKGEAAAERPGE SEQ ID NO. 11 peptide 12 AQFSKTAAKGEAAAERPGEASEQ ID NO. 12 peptide 13 QFSKTAAKGEAAAERPGEAA SEQ ID NO. 13 peptide 14FSKTAAKGEAAAERPGEAAV SEQ ID NO. 14 peptide 15 SKTAAKGEAAAERPGEAAVASEQ ID NO. 15 peptide 16 GAQFSKTAAKGEAAAERPG SEQ ID NO. 16 peptide 17AQFSKTAAKGEAAAERPGE SEQ ID NO. 17 peptide 18 QFSKTAAKGEAAAERPGEASEQ ID NO. 18 peptide 19 FSKTAAKGEAAAERPGEAA SEQ ID NO. 19 peptide 20SKTAAKGEAAAERPGEAAV SEQ ID NO. 20 peptide 21 KTAAKGEAAAERPGEAAVASEQ ID NO. 21 peptide 22 GAQFSKTAAKGEAAAERP SEQ ID NO. 22 peptide 23AQFSKTAAKGEAAAERPG SEQ ID NO. 23 peptide 24 QFSKTAAKGEAAAERPGESEQ ID NO. 24 peptide 25 FSKTAAKGEAAAERPGEA SEQ ID NO. 25 peptide 26SKTAAKGEAAAERPGEAA SEQ ID NO. 26 peptide 27 KTAAKGEAAAERPGEAAVSEQ ID NO. 27 peptide 28 TAAKGEAAAERPGEAAVA SEQ ID NO. 28 peptide 29GAQFSKTAAKGEAAAER SEQ ID NO. 29 peptide 30 AQFSKTAAKGEAAAERPSEQ ID NO. 30 peptide 31 QFSKTAAKGEAAAERPG SEQ ID NO. 31 peptide 32FSKTAAKGEAAAERPGE SEQ ID NO. 32 peptide 33 SKTAAKGEAAAERPGEASEQ ID NO. 33 peptide 34 KTAAKGEAAAERPGEAA SEQ ID NO. 34 peptide 35TAAKGEAAAERPGEAAV SEQ ID NO. 35 peptide 36 AAKGEAAAERPGEAAVASEQ ID NO. 36 peptide 37 GAQFSKTAAKGEAAAE SEQ ID NO. 37 peptide 38AQFSKTAAKGEAAAER SEQ ID NO. 38 peptide 39 QFSKTAAKGEAAAERP SEQ ID NO. 39peptide 40 FSKTAAKGEAAAERPG SEQ ID NO. 40 peptide 41 SKTAAKGEAAAERPGESEQ ID NO. 41 peptide 42 KTAAKGEAAAERPGEA SEQ ID NO. 42 peptide 43TAAKGEAAAERPGEAA SEQ ID NO. 43 peptide 44 AAKGEAAAERPGEAAV SEQ ID NO. 44peptide 45 AKGEAAAERPGEAAVA SEQ ID NO. 45 peptide 46 GAQFSKTAAKGEAAASEQ ID NO. 46 peptide 47 AQFSKTAAKGEAAAE SEQ ID NO. 47 peptide 48QFSKTAAKGEAAAER SEQ ID NO. 48 peptide 49 FSKTAAKGEAAAERP SEQ ID NO. 49peptide 50 SKTAAKGEAAAERPG SEQ ID NO. 50 peptide 51 KTAAKGEAAAERPGESEQ ID NO. 51 peptide 52 TAAKGEAAAERPGEA SEQ ID NO. 52 peptide 53AAKGEAAAERPGEAA SEQ ID NO. 53 peptide 54 AKGEAAAERPGEAAV SEQ ID NO. 54peptide 55 KGEAAAERPGEAAVA SEQ ID NO. 55 peptide 56 GAQFSKTAAKGEAASEQ ID NO. 56 peptide 57 AQFSKTAAKGEAAA SEQ ID NO. 57 peptide 58QFSKTAAKGEAAAE SEQ ID NO. 58 peptide 59 FSKTAAKGEAAAER SEQ ID NO. 59peptide 60 SKTAAKGEAAAERP SEQ ID NO. 60 peptide 61 KTAAKGEAAAERPGSEQ ID NO. 61 peptide 62 TAAKGEAAAERPGE SEQ ID NO. 62 peptide 63AAKGEAAAERPGEA SEQ ID NO. 63 peptide 64 AKGEAAAERPGEAA SEQ ID NO. 64peptide 65 KGEAAAERPGEAAV SEQ ID NO. 65 peptide 66 GEAAAERPGEAAVASEQ ID NO. 66 peptide 67 GAQFSKTAAKGEA SEQ ID NO. 67 peptide 68AQFSKTAAKGEAA SEQ ID NO. 68 peptide 69 QFSKTAAKGEAAA SEQ ID NO. 69peptide 70 SKTAAKGEAAAE SEQ ID NO. 70 peptide 71 SKTAAKGEAAAERSEQ ID NO. 71 peptide 72 KTAAKGEAAAERP SEQ ID NO. 72 peptide 73TAAKGEAAAERPG SEQ ID NO. 73 peptide 74 AAKGEAAAERPGE SEQ ID NO. 74peptide 75 AKGEAAAERPGEA SEQ ID NO. 75 peptide 76 KGEAAAERPGEAASEQ ID NO. 76 peptide 77 GEAAAERPGEAAV SEQ ID NO. 77 peptide 78EAAAERPGEAAVA SEQ ID NO. 78 peptide 79 GAQFSKTAAKGE SEQ ID NO. 79peptide 80 AQFSKTAAKGEA SEQ ID NO. 80 peptide 81 QFSKTAAKGEAASEQ ID NO. 81 peptide 82 FSKTAAKGEAAA SEQ ID NO. 82 peptide 83SKTAAKGEAAAE SEQ ID NO. 83 peptide 84 KTAAKGEAAAER SEQ ID NO. 84peptide 85 TAAKGEAAAERP SEQ ID NO. 85 peptide 86 AAKGEAAAERPGSEQ ID NO. 86 peptide 87 AKGEAAAERPGE SEQ ID NO. 87 peptide 88KGEAAAERPGEA SEQ ID NO. 88 peptide 89 GEAAAERPGEAA SEQ ID NO. 89peptide 90 EAAAERPGEAAV SEQ ID NO. 90 peptide 91 AAAERPGEAAVASEQ ID NO. 91 peptide 92 GAQFSKTAAKG SEQ ID NO. 92 peptide 93AQFSKTAAKGE SEQ ID NO. 93 peptide 94 QFSKTAAKGEA SEQ ID NO. 94peptide 95 FSKTAAKGEAA SEQ ID NO. 95 peptide 96 SKTAAKGEAAASEQ ID NO. 96 peptide 97 KTAAKGEAAAE SEQ ID NO. 97 peptide 98TAAKGEAAAER SEQ ID NO. 98 peptide 99 AAKGEAAAERP SEQ ID NO. 99peptide 100 AKGEAAAERPG SEQ ID NO. 100 peptide 101 KGEAAAERPGESEQ ID NO. 101 peptide 102 GEAAAERPGEA SEQ ID NO. 102 peptide 103EAAAERPGEAA SEQ ID NO. 103 peptide 104 AAAERPGEAAV SEQ ID NO. 104peptide 105 AAERPGEAAVA SEQ ID NO. 105 peptide 106 GAQFSKTAAKSEQ ID NO. 106 peptide 107 AQFSKTAAKG SEQ ID NO. 107 peptide 108QFSKTAAKGE SEQ ID NO. 108 peptide 109 FSKTAAKGEA SEQ ID NO. 109peptide 110 SKTAAKGEAA SEQ ID NO. 110 peptide 111 KTAAKGEAAASEQ ID NO. 111 peptide 112 TAAKGEAAAE SEQ ID NO. 112 peptide 113AAKGEAAAER SEQ ID NO. 113 peptide 114 AKGEAAAERP SEQ ID NO. 114peptide 115 KGEAAAERPG SEQ ID NO. 115 peptide 116 GEAAAERPGESEQ ID NO. 116 peptide 117 EAAAERPGEA SEQ ID NO. 117 peptide 118AAAERPGEAA SEQ ID NO. 118 peptide 119 AAERPGEAAV SEQ ID NO. 119peptide 120 AERPGEAAVA SEQ ID NO. 120 peptide 121 GAQFSKTAASEQ ID NO. 121 peptide 122 AQFSKTAAK SEQ ID NO. 122 peptide 123QFSKTAAKG SEQ ID NO. 123 peptide 124 FSKTAAKGE SEQ ID NO. 124peptide 125 SKTAAKGEA SEQ ID NO. 125 peptide 126 KTAAKGEAASEQ ID NO. 126 peptide 127 TAAKGEAAA SEQ ID NO. 127 peptide 128AAKGEAAAE SEQ ID NO. 128 peptide 129 AKGEAAAER SEQ ID NO. 129peptide 130 KGEAAAERP SEQ ID NO. 130 peptide 131 GEAAAERPGSEQ ID NO. 131 peptide 132 EAAAERPGE SEQ ID NO. 132 peptide 133AAAERPGEA SEQ ID NO. 133 peptide 134 AAERPGEAA SEQ ID NO. 134peptide 135 AERPGEAAV SEQ ID NO. 135 peptide 136 ERPGEAAVASEQ ID NO. 136 peptide 137 GAQFSKTA SEQ ID NO. 137 peptide 138 AQFSKTAASEQ ID NO. 138 peptide 139 QFSKTAAK SEQ ID NO. 139 peptide 140 FSKTAAKGSEQ ID NO. 140 peptide 141 SKTAAKGE SEQ ID NO. 141 peptide 142 KTAAKGEASEQ ID NO. 142 peptide 143 TAAKGEAA SEQ ID NO. 143 peptide 144 AAKGEAAASEQ ID NO. 144 peptide 145 AKGEAAAE SEQ ID NO. 145 peptide 146 KGEAAAERSEQ ID NO. 146 peptide 147 GEAAAERP SEQ ID NO. 147 peptide 148 EAAAERPGSEQ ID NO. 148 peptide 149 AAAERPGE SEQ ID NO. 149 peptide 150 AAERPGEASEQ ID NO. 150 peptide 151 AERPGEAA SEQ ID NO. 151 peptide 152 ERPGEAAVSEQ ID NO. 152 peptide 153 RPGEAAVA SEQ ID NO. 153 peptide 154 GAQFSKTSEQ ID NO. 154 peptide 155 AQFSKTA SEQ ID NO. 155 peptide 156 QFSKTAASEQ ID NO. 156 peptide 157 FSKTAAK SEQ ID NO. 157 peptide 158 SKTAAKGSEQ ID NO. 158 peptide 159 KTAAKGE SEQ ID NO. 159 peptide 160 TAAKGEASEQ ID NO. 160 peptide 161 AAKGEAA SEQ ID NO. 161 peptide 162 AKGEAAASEQ ID NO. 162 peptide 163 KGEAAAE SEQ ID NO. 163 peptide 164 GEAAAERSEQ ID NO. 164 peptide 165 EAAAERP SEQ ID NO. 165 peptide 166 AAAERPGSEQ ID NO. 166 peptide 167 AAERPGE SEQ ID NO. 167 peptide 168 AERPGEASEQ ID NO. 168 peptide 169 ERPGEAA SEQ ID NO. 169 peptide 170 RPGEAAVSEQ ID NO. 170 peptide 171 PGEAAVA SEQ ID NO. 171 peptide 172 GAQFSKSEQ ID NO. 172 peptide 173 AQFSKT SEQ ID NO. 173 peptide 174 QFSKTASEQ ID NO. 174 peptide 175 FSKTAA SEQ ID NO. 175 peptide 176 SKTAAKSEQ ID NO. 176 peptide 177 KTAAKG SEQ ID NO. 177 peptide 178 TAAKGESEQ ID NO. 178 peptide 179 AAKGEA SEQ ID NO. 179 peptide 180 AKGEAASEQ ID NO. 180 peptide 181 KGEAAA SEQ ID NO. 181 peptide 182 GEAAAESEQ ID NO. 182 peptide 183 EAAAER SEQ ID NO. 183 peptide 184 AAAERPSEQ ID NO. 184 peptide 185 AAERPG SEQ ID NO. 185 peptide 186 AERPGESEQ ID NO. 186 peptide 187 ERPGEA SEQ ID NO. 187 peptide 188 RPGEAASEQ ID NO. 188 peptide 189 PGEAAV SEQ ID NO. 189 peptide 190 GEAAVASEQ ID NO. 190 peptide 191 GAQFS SEQ ID NO. 191 peptide 192 AQFSKSEQ ID NO. 192 peptide 193 QFSKT SEQ ID NO. 193 peptide 194 FSKTASEQ ID NO. 194 peptide 195 SKTAA SEQ ID NO. 195 peptide 196 KTAAKSEQ ID NO. 196 peptide 197 TAAKG SEQ ID NO. 197 peptide 198 AAKGESEQ ID NO. 198 peptide 199 AKGEA SEQ ID NO. 199 peptide 200 KGEAASEQ ID NO. 200 peptide 201 GEAAA SEQ ID NO. 201 peptide 202 EAAAESEQ ID NO. 202 peptide 203 AAAER SEQ ID NO. 203 peptide 204 AAERPSEQ ID NO. 204 peptide 205 AERPG SEQ ID NO. 205 peptide 206 ERPGESEQ ID NO. 206 peptide 207 RPGEA SEQ ID NO. 207 peptide 208 PGEAASEQ ID NO. 208 peptide 209 GEAAV SEQ ID NO. 209 peptide 210 EAAVASEQ ID NO. 210 peptide 211 GAQF SEQ ID NO. 211 peptide 212 AQFSSEQ ID NO. 212 peptide 213 QFSK SEQ ID NO. 213 peptide 214 FSKTSEQ ID NO. 214 peptide 215 SKTA SEQ ID NO. 215 peptide 216 KTAASEQ ID NO. 216 peptide 217 TAAK SEQ ID NO. 217 peptide 218 AAKGSEQ ID NO. 218 peptide 219 AKGE SEQ ID NO. 219 peptide 220 KGEASEQ ID NO. 220 peptide 221 GEAA SEQ ID NO. 221 peptide 222 EAAASEQ ID NO. 222 peptide 223 AAAE SEQ ID NO. 223 peptide 224 AAERSEQ ID NO. 224 peptide 225 AERP SEQ ID NO. 225 peptide 226 ERPGSEQ ID NO. 226 peptide 227 RPGE SEQ ID NO. 227 peptide 228 PGEASEQ ID NO. 228 peptide 229 GEAA SEQ ID NO. 229 peptide 230 EAAVSEQ ID NO. 230 peptide 231 AAVA SEQ ID NO. 231 peptide 232GTAPAAEGAGAEVKRASAEAKQAF SEQ ID NO. 232 peptide 233 GKQFSKTAAKGESEQ ID NO. 233 peptide 234 GAQFSKTKAKGE SEQ ID NO. 234 peptide 235GKQFSKTKAKGE SEQ ID NO. 235 peptide 236 GAQASKTAAK SEQ ID NO. 236peptide 237 GAQASKTAAKGE SEQ ID NO. 237 peptide 238 GAEFSKTAAKGESEQ ID NO. 238 peptide 239 GAQFSKTAAAGE SEQ ID NO. 239 peptide 240GAQFSKTAAKAE SEQ ID NO. 240 peptide 241 GAQFSKTAAKGA SEQ ID NO. 241peptide 242 AAQFSKTAAK SEQ ID NO. 242 peptide 243 GAAFSKTAAKSEQ ID NO. 243 peptide 244 GAQFAKTAAK SEQ ID NO. 244 peptide 245GAQFSATAAK SEQ ID NO. 245 peptide 246 KAATKSFQAG SEQ ID NO. 246peptide 247 GAQFSKAAAK SEQ ID NO. 247 peptide 248 GAQFSKTAAASEQ ID NO. 248 peptide 249 GAQFSATAAA SEQ ID NO. 249 peptide 250GAQASKTA SEQ ID NO. 250 peptide 251 AAGE SEQ ID NO. 251 peptide 252GKASQFAKTA SEQ ID NO. 252

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made without departingfrom the spirit and scope of the invention.

1-24. (canceled)
 25. A method of inhibiting mucin hypersecretion in amammal, the method comprising administering to the mammal a mucinhypersecretion-inhibiting amount of a N-terminal- and/orC-terminal-chemically modified peptide or a pharmaceutical compositioncomprising said modified peptide, which peptide consists of an aminoacid sequence selected from the group consisting of: (a) a contiguous 4to 6 amino acid segment of a reference sequence,GAQFSKTAAKGEAAAERPGEAAVA (SEQ ID NO:1), wherein the amino acid segmentis selected from the group consisting of SEQ ID NO: 176 to SEQ ID NO:180, SEQ ID NO: 196 to SEQ ID NO: 199, SEQ ID NO: 217 and SEQ ID NO:219; wherein (i) the C-terminal amino acid of said chemically modifiedpeptide is chemically modified and the N-terminal amino acid of thepeptide is alkylated or acylated at the N-terminal amino group; or (ii)the C-terminal amino acid of said chemically modified peptide is notchemically modified and the N-terminal amino acid of the peptide isalkylated or acylated at the N-terminal amino group and is notmyristoylated; with the proviso that said chemically modified peptidecontains no more than 6 amino acids; and wherein said chemicallymodified peptide has a mucin hypersecretion-inhibiting effect whenadministered to a mammal in a mucin hypersecretion-inhibiting amount.26. The method of claim 25, wherein the amino acid sequence of thepeptide includes the contiguous residues AKGE (SEQ ID NO: 219) of thereference sequence.
 27. (canceled)
 28. The method of claim 25, whereinthe N-terminal amino acid of the peptide is acetylated at the N-terminalamino group.
 29. The method of claim 25, wherein said peptide isacetyl-PEPTIDE 179, acetyl-PEPTIDE 219, or acetyl-PEPTIDE 219-NH₂. 30.(canceled)
 31. The method of claim 25, wherein the amino acid sequenceis selected from the group consisting of the amino acid sequence of SEQID NO: 178 to SEQ ID NO: 180, SEQ ID NO: 198, SEQ ID NO: 199 and SEQ IDNO:
 219. 32. The method of claim 25, wherein the N-terminal amino acidis alkylated at the N-terminal amine with a C1 to C24 aliphatic alkylgroup, a linear 2-(C1 to C24 aliphatic alkyl)oxyethyl group, or anomega-methoxy-poly(ethyleneoxy)n-ethyl group, where n is from 0 to 10.33. The method of claim 25, wherein the N-terminal amino acid isacylated at the N-terminal amine with trifluoroacetic acid, benzoicacid, or a C₁ to C₂₄ aliphatic carboxylic acid.
 34. The method of claim25, wherein the C-terminal amino acid of the peptide is amidated oresterified at the C-terminal carboxyl group.
 35. The method of claim 25,wherein the C-terminal amino acid is amidated at the C-terminal carboxylgroup with ammonia, a C1 to C24 aliphatic alkyl amine, ahydroxyl-substituted C2 to C24 aliphatic alkyl amine, a linear 2-(C1 toC24 aliphatic alkyl)oxyethylamine group, or anomega-methoxy-poly(ethyleneoxy)_(n)-ethylamine group, where n is from 0to
 10. 36. The method of claim 25, wherein the C-terminal amino acid isesterified at the C-terminal carboxyl group with a C1 to C24 aliphaticalkyl alcohol or a 2-(omega-methoxy-poly(ethyleneoxy)_(n))-ethanolgroup, where n is from of 0 to
 10. 37-39. (canceled)
 40. The method ofclaim 25, wherein the peptide exhibits at least one of the properties of(a) greater mucin hypersecretion-inhibiting effect on a mammal than SEQID NO:1, wherein the N-terminal amino acid is myristoylated, whenadministered to said mammal at equal concentrations or (b) greateraqueous solubility than SEQ ID NO:1, wherein the N-terminal amino acidis myristoylated, at equal concentrations in the same liquid.
 41. Themethod of claim 25, wherein the mucin hypersecretion in the mammal iscaused by a pulmonary disease in the mammal.
 42. The method of claim 41,wherein the pulmonary disease is selected from the group consisting ofasthma, chronic bronchitis, chronic obstructive pulmonary disease(COPD), and cystic fibrosis.
 43. The method of claim 25, wherein themucin hypersecretion in the mammal is caused by a disease or conditionselected from the group consisting of an inflammatory disease, a viralor bacterial airway disease, common cold, rhinitis, acute or chronicbronchitis, pneumonia, kennel cough, allergic conditions,bronchiectasis, emphysema, and bronchial asthma.
 44. The method of claim25, wherein the pharmaceutical composition comprises a pharmaceuticallyacceptable carrier.
 45. The method of claim 25, wherein thepharmaceutical composition is suitable for pulmonary administration.46.-52. (canceled)